Detecting nucleic acid

ABSTRACT

This document provides methods and materials for detecting target nucleic acid. For example, methods and materials for detecting the presence or absence of target nucleic acid, methods and materials for detecting the amount of target nucleic acid present within a sample, kits for detecting the presence or absence of target nucleic acid, kits for detecting the amount of target nucleic acid present within a sample, and methods for making such kits are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/535,017, filed Aug. 4, 2009, which claims the benefit of priority ofU.S. Provisional Application Ser. No. 61/089,392, filed Aug. 15, 2008,and U.S. Provisional Application Ser. No. 61/166,843, filed Apr. 6,2009. The disclosures of the prior applications are considered part of(and are incorporated by reference in) the disclosure of thisapplication.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in detectingnucleic acid. For example, this document relates to methods andmaterials involved in using an enzymatic amplification cascade ofrestriction endonucleases to detect nucleic acid.

2. Background

The polymerase chain reaction (PCR) is a technique commonly used toamplify and detect nucleic acid. For example, real-time PCR can be usedto amplify and detect small amounts of template DNA present in a sample.Typically, PCR involves adding a nucleic acid primer pair and aheat-stable DNA polymerase, such as Taq polymerase, to a sample believedto contain a targeted nucleic acid to be amplified. Subjecting thesample containing the primer pair and polymerase to a thermal cyclingprocess sets in motion a chain reaction in which the targeted DNAtemplate located between the primers is exponentially amplified. Thepresence of this amplified template can be detected using techniquessuch as gel electrophoresis or the amount of amplified template can beassessed using techniques such as those involving the use offluorescently labeled probes.

SUMMARY

This document provides methods and materials for detecting targetnucleic acid. For example, this document provides methods and materialsfor detecting the presence or absence of target nucleic acid within asample, methods and materials for detecting the amount of target nucleicacid present within a sample, kits for detecting the presence or absenceof target nucleic acid within a sample, kits for detecting the amount oftarget nucleic acid present within a sample, and methods for making suchkits. In general, the methods and materials provided herein can includeperforming an enzymatic amplification cascade of restrictionendonucleases as described herein to detect target nucleic acid in amanner that is rapid, inexpensive, sensitive, and specific. In somecases, the methods and materials provided herein can be used in additionto or can replace current PCR-based nucleic acid detection approaches.

The methods and materials provided herein can allow clinicians, medicalprofessionals, laboratory personnel, and researchers to detect any typeof target nucleic acid. For example, the methods and materials providedherein can be used in genotyping applications to detect nucleic acidmutations (e.g., single nucleotide polymorphisms (SNPs)), genomerearrangements, and genome epigenetic events (e.g., DNA methylationevents), can be used in diagnostic or prognostic applications to detectviruses or microorganisms (e.g., bacteria, fungi, and protozoa), can beused in gene expression applications to detect mRNA levels withinparticular cell types, and can be used in forensic applications tocompare the identity between samples or to assess a sample's origin. Insome cases, the methods and materials provided herein can be used byindividuals outside the medical/biotechnology profession to detecttarget nucleic acid. For example, a kit provided herein for detectingthe presence of bacteria (e.g., E. coli) can be designed for use as ahome detection kit such that a user can detect the presence or absenceof E. coli nucleic acid within a biological sample (e.g., blood sample,mucus sample, or saliva sample) obtained from the user.

In general, one aspect of this document features a method for assessinga sample for target nucleic acid. The method comprises, or consistsessentially of: (a) contacting the sample with a probe nucleic acidcomprising an amplifying restriction endonuclease and a nucleotidesequence complementary to a sequence of the target nucleic acid underconditions wherein, if the target nucleic acid is present in the sample,at least a portion of the target nucleic acid hybridizes to at least aportion of the probe nucleic acid to form a double-stranded portion ofnucleic acid comprising a restriction endonuclease cut site, (b)contacting the double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site under conditions wherein the recognition restrictionendonuclease cleaves the double-stranded portion of nucleic acid at therestriction endonuclease cut site, thereby separating a portion of theprobe nucleic acid comprising the amplifying restriction endonucleasefrom at least another portion of the probe nucleic acid, (c) contactingthe portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease with a reporter nucleic acid comprising adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the amplifying restriction endonuclease underconditions wherein the amplifying restriction endonuclease cleaves thereporter nucleic acid at the restriction endonuclease cut site of theamplifying restriction endonuclease, thereby separating a portion of thereporter nucleic acid from at least another portion of the reporternucleic acid, and (d) determining the presence or absence of the portionof the reporter nucleic acid, wherein the presence of the portion of thereporter nucleic acid indicates that the sample contains the targetnucleic acid, and wherein the absence of the portion of the reporternucleic acid indicates that the sample does not contain the targetnucleic acid. The probe nucleic acid can be single-stranded probenucleic acid. The probe nucleic acid can be attached to a solid support.The probe nucleic acid can be directly attached to a solid support. Theportion of the probe nucleic acid comprising the amplifying restrictionendonuclease can be released from the solid support via step (b). Step(a) and step (b) can be performed in the same compartment. Step (a) andstep (b) can be performed in a first compartment, and step (c) can beperformed in a second compartment. Step (a) and step (b) can beperformed by adding the sample to a compartment comprising the probenucleic acid and the recognition restriction endonuclease. The probenucleic acid can comprise (i) a single-stranded portion comprising thenucleotide sequence complementary to the sequence of the target nucleicacid and (ii) a double-stranded portion. The probe nucleic acid cancomprise a first nucleic acid strand, which can comprise the nucleotidesequence complementary to the sequence of the target nucleic acid,hybridized to a second nucleic acid strand comprising the amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. A portion of the second nucleic acid strandcan be hybridized with the first nucleic acid strand to form thedouble-stranded portion. The portion of the probe nucleic acidcomprising the amplifying restriction endonuclease that is separatedfrom the at least another portion of the probe nucleic acid in step (b)can comprise a portion of the first nucleic acid strand and all of thesecond strand. The portion of the probe nucleic acid comprising theamplifying restriction endonuclease that is separated from the at leastanother portion of the probe nucleic acid in step (b) can comprise atleast a portion of the target nucleic acid. The method can compriseusing a plurality of the probe nucleic acid in step (a). The method cancomprise using a plurality of the reporter nucleic acid in step (c). Thereporter nucleic acid in step (c) can be in molar excess of the portionof the probe nucleic acid comprising the amplifying restrictionendonuclease from the step (b). The number of molecules of the portionof the probe nucleic acid comprising the amplifying restrictionendonuclease that is separated from the at least another portion of theprobe nucleic acid in step (b) can be in an essentially linearrelationship to the number of molecules of the target nucleic acidpresent in the sample. The reporter nucleic acid can be attached to asolid support. The reporter nucleic acid can be directly attached to asolid support. The reporter nucleic acid can comprise a single-strandedportion of nucleic acid. The reporter nucleic acid can comprise a label.The label can be a fluorescent label, a radioactive label, an enzymelabel, or a redox label. The portion of the reporter nucleic acid thatis separated from the at least another portion of the reporter nucleicacid can comprise the label. The reporter nucleic acid can comprise afirst nucleic acid strand, which can comprise the label, hybridized to asecond nucleic acid strand. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. A portion of the first nucleicacid strand can hybridize with the second nucleic acid strand to formthe double-stranded portion of nucleic acid comprising the restrictionendonuclease cut site of the amplifying restriction endonuclease. Thereporter nucleic acid can comprise a third nucleic acid strand. Thethird nucleic acid strand can hybridize with the second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the amplifying restrictionendonuclease. The reporter nucleic acid can be attached to a solidsupport, and the portion of the reporter nucleic acid that is separatedfrom the at least another portion of the reporter nucleic acid and thatcomprises the label can be released from the solid support via step (c).Determining step (d) can comprise detecting the label. The label can bea fluorescent label, and determining step (d) can comprise detecting thefluorescent label. Determining step (d) can comprise detecting theportion of the reporter nucleic acid separated from the at least anotherportion of the reporter nucleic acid using a capillary electrophoresistechnique. Steps (a), (b), and (c) can be performed without nucleic acidamplification. Steps (a), (b), (c), and (d) can be performed withoutnucleic acid amplification. The determining step can comprisedetermining the amount of the target nucleic acid present within thesample.

In another aspect, this document features a method for assessing asample for target nucleic acid. The method comprises, or consistsessentially of, (a) contacting the sample with a probe nucleic acidcomprising an initial amplifying restriction endonuclease and anucleotide sequence complementary to a sequence of the target nucleicacid under conditions wherein, if the target nucleic acid is present inthe sample, at least a portion of the target nucleic acid hybridizes toat least a portion of the probe nucleic acid to form a double-strandedportion of nucleic acid comprising a restriction endonuclease cut site,(b) contacting the double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site under conditions wherein the recognition restrictionendonuclease cleaves the double-stranded portion of nucleic acid at therestriction endonuclease cut site, thereby separating a portion of theprobe nucleic acid comprising the initial amplifying restrictionendonuclease from at least another portion of the probe nucleic acid,(c) contacting the portion of the probe nucleic acid comprising theinitial amplifying restriction endonuclease with a first signalexpansion nucleic acid comprising a secondary amplifying restrictionendonuclease and a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site of the initial amplifying restrictionendonuclease under conditions wherein the initial amplifying restrictionendonuclease cleaves the first signal expansion nucleic acid at therestriction endonuclease cut site of the initial amplifying restrictionendonuclease, thereby separating a portion of the first signal expansionnucleic acid comprising the secondary amplifying restrictionendonuclease from at least another portion of the first nucleic acid,(d) contacting the portion of the first signal expansion nucleic acidcomprising the secondary amplifying restriction endonuclease with asecond signal expansion nucleic acid comprising the initial amplifyingrestriction endonuclease and a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site of the secondaryamplifying restriction endonuclease under conditions wherein thesecondary amplifying restriction endonuclease cleaves the second signalexpansion nucleic acid at the restriction endonuclease cut site of thesecondary amplifying restriction endonuclease, thereby separating aportion of the second signal expansion nucleic acid comprising theinitial amplifying restriction endonuclease from at least anotherportion of the second signal expansion nucleic acid, (e) contacting (i)the portion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease, (ii) the portion of the second signalexpansion nucleic acid comprising the initial amplifying restrictionendonuclease, (iii) the portion of the first signal expansion nucleicacid comprising the secondary amplifying restriction endonuclease, or(iv) any combination thereof with a reporter nucleic acid comprising adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the initial amplifying restriction endonucleaseand/or a restriction endonuclease cut site of the secondary amplifyingrestriction endonuclease under conditions wherein the initial amplifyingrestriction endonuclease cleaves the reporter nucleic acid at therestriction endonuclease cut site of the initial amplifying restrictionendonuclease, thereby separating a portion of the reporter nucleic acidfrom at least another portion of the reporter nucleic acid, and (f)determining the presence or absence of the portion of the reporternucleic acid, wherein the presence of the portion of the reporternucleic acid indicates that the sample contains the target nucleic acid,and wherein the absence of the portion of the reporter nucleic acidindicates that the sample does not contain the target nucleic acid. Theprobe nucleic acid can be single-stranded probe nucleic acid. The probenucleic acid can be attached to a solid support. The probe nucleic acidcan be directly attached to a solid support. The portion of the probenucleic acid comprising the initial amplifying restriction endonucleasecan be released from the solid support via step (b). Step (a) and step(b) can be performed in the same compartment. Step (c) and step (d) canbe performed in the same compartment. Step (a) and step (b) can beperformed in a first compartment, and step (c) and step (d) can beperformed in a second compartment. Step (a) and step (b) can beperformed by adding the sample to a compartment comprising the probenucleic acid and the recognition restriction endonuclease. Step (c) andstep (d) can be performed by adding the portion of the probe nucleicacid comprising the initial amplifying restriction endonuclease to acompartment comprising the first signal expansion nucleic acid and thesecond signal expansion nucleic acid. The probe nucleic acid cancomprise (i) a single-stranded portion comprising the nucleotidesequence complementary to the sequence of the target nucleic acid and(ii) a double-stranded portion. The probe nucleic acid can comprise afirst nucleic acid strand, which can comprise the nucleotide sequencecomplementary to the sequence of the target nucleic acid, hybridized toa second nucleic acid strand comprising the initial amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. A portion of the second nucleic acid strandcan be hybridized with the first nucleic acid strand to form thedouble-stranded portion. The portion of the probe nucleic acidcomprising the initial amplifying restriction endonuclease that isseparated from the at least another portion of the probe nucleic acid instep (b) can comprise a portion of the first nucleic acid strand and allof the second strand. The portion of the probe nucleic acid comprisingthe initial amplifying restriction endonuclease that is separated fromthe at least another portion of the probe nucleic acid in step (b) cancomprise at least a portion of the target nucleic acid. The method cancomprise using a plurality of the probe nucleic acid in step (a). Themethod can comprise using a plurality of the reporter nucleic acid instep (e). The reporter nucleic acid in step (e) can be in molar excessof the portion of the probe nucleic acid comprising the initialamplifying restriction endonuclease from step (b). The number ofmolecules of the portion of the probe nucleic acid comprising theinitial amplifying restriction endonuclease that is separated from theat least another portion of the probe nucleic acid in step (b) can be inan essentially linear relationship to the number of molecules of thetarget nucleic acid present in the sample. The first signal expansionnucleic acid and the second signal expansion nucleic acid can beattached to a solid support. The first signal expansion nucleic acid andthe second signal expansion nucleic acid can be directly attached to asolid support. The first signal expansion nucleic acid and the secondsignal expansion nucleic acid can be attached to a solid support in thesame compartment. The portion of the first signal expansion nucleic acidcomprising the secondary amplifying restriction endonuclease can bereleased from the solid support via step (c). The portion of the secondsignal expansion nucleic acid comprising the initial amplifyingrestriction endonuclease can be released from the solid support via step(d). The first signal expansion nucleic acid can comprise a firstnucleic acid strand, which can comprise the secondary amplifyingrestriction endonuclease, hybridized to a second nucleic acid strand toform the double-stranded portion of nucleic acid comprising therestriction endonuclease cut site of the initial amplifying restrictionendonuclease. The first nucleic acid strand can be attached to a solidsupport. The first nucleic acid strand can be directly attached to asolid support. The second nucleic acid strand can be attached to a solidsupport. The second nucleic acid strand can be directly attached to asolid support. The second signal expansion nucleic acid can comprise afirst nucleic acid strand, which can comprise the initial amplifyingrestriction endonuclease, hybridized to a second nucleic acid strand toform the double-stranded portion of nucleic acid comprising therestriction endonuclease cut site of the secondary amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The reporter nucleic acid can beattached to a solid support. The reporter nucleic acid can be directlyattached to a solid support. The reporter nucleic acid can comprise asingle-stranded portion of nucleic acid. The reporter nucleic acid cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The portion of the reporternucleic acid that is separated from the at least another portion of thereporter nucleic acid can comprise the label. The reporter nucleic acidcan comprise a first nucleic acid strand, which can comprise the label,hybridized to a second nucleic acid strand. The second nucleic acidstrand can be attached to a solid support. The second nucleic acidstrand can be directly attached to a solid support. A portion of thefirst nucleic acid strand can hybridize with the second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the initial amplifyingrestriction endonuclease. The reporter nucleic acid can comprise a thirdnucleic acid strand. The third nucleic acid strand can be hybridizedwith the second nucleic acid strand to form the double-stranded portionof nucleic acid comprising the restriction endonuclease cut site of theinitial amplifying restriction endonuclease. The reporter nucleic acidcan be attached to a solid support, and the portion of the reporternucleic acid that is separated from the at least another portion of thereporter nucleic acid and that comprises the label can be released fromthe solid support via step (e). Determining step (f) can comprisedetecting the label. The label can be a fluorescent label, anddetermining step (f) can comprise detecting the fluorescent label.Determining step (f) can comprise detecting the portion of the reporternucleic acid separated from the at least another portion of the reporternucleic acid using a capillary electrophoresis technique. Steps (a),(b), (c), (d), and (e) can be performed without nucleic acidamplification. Steps (a), (b), (c), (d), (e), and (f) can be performedwithout nucleic acid amplification. The determining step can comprisedetermining the amount of the target nucleic acid present within thesample.

In another aspect, this document features a method for assessing asample for target nucleic acid. The method comprises, or consistsessentially of, (a) contacting the sample with a probe nucleic acidcomprising an initial amplifying restriction endonuclease and anucleotide sequence complementary to a sequence of the target nucleicacid under conditions wherein, if the target nucleic acid is present inthe sample, at least a portion of the target nucleic acid hybridizes toat least a portion of the probe nucleic acid to form a double-strandedportion of nucleic acid comprising a restriction endonuclease cut site,(b) contacting the double-stranded portion of nucleic acid with arecognition restriction endonuclease having the ability to cut thedouble-stranded portion of nucleic acid at the restriction endonucleasecut site under conditions wherein the recognition restrictionendonuclease cleaves the double-stranded portion of nucleic acid at therestriction endonuclease cut site, thereby separating a portion of theprobe nucleic acid comprising the initial amplifying restrictionendonuclease from at least another portion of the probe nucleic acid,(c) contacting the portion of the probe nucleic acid comprising theinitial amplifying restriction endonuclease with a first reporternucleic acid (or a first signal expansion nucleic acid) comprising asecondary amplifying restriction endonuclease and a double-strandedportion of nucleic acid comprising a restriction endonuclease cut siteof the initial amplifying restriction endonuclease under conditionswherein the initial amplifying restriction endonuclease cleaves thefirst reporter nucleic acid at the restriction endonuclease cut site ofthe initial amplifying restriction endonuclease, thereby separating aportion of the first nucleic acid comprising the secondary amplifyingrestriction endonuclease from at least another portion of the firstnucleic acid, (d) contacting the portion of the first reporter nucleicacid comprising the secondary amplifying restriction endonuclease with asecond reporter nucleic acid (or a second signal expansion nucleic acid)comprising the initial amplifying restriction endonuclease and adouble-stranded portion of nucleic acid comprising a restrictionendonuclease cut site of the secondary amplifying restrictionendonuclease under conditions wherein the initial amplifying restrictionendonuclease cleaves the second nucleic acid at the restrictionendonuclease cut site of the secondary amplifying restrictionendonuclease, thereby separating a portion of the second nucleic acidcomprising the initial amplifying restriction endonuclease from at leastanother portion of the second nucleic acid, and (e) determining thepresence or absence of the portion of the first reporter nucleic acid,the second reporter nucleic acid, or both the first reporter nucleicacid and the second reporter nucleic acid, wherein the presenceindicates that the sample contains the target nucleic acid, and whereinthe absence indicates that the sample does not contain the targetnucleic acid. The probe nucleic acid can be single-stranded probenucleic acid. The probe nucleic acid can be attached to a solid support.The probe nucleic acid can be directly attached to a solid support. Theportion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease can be released from the solid support via step(b). Step (a) and step (b) can be performed in the same compartment.Step (c) and step (d) can be performed in the same compartment. Step (a)and step (b) can be performed in a first compartment, and step (c) andstep (d) can be performed in a second compartment. Step (a) and step (b)can be performed by adding the sample to a compartment comprising theprobe nucleic acid and the recognition restriction endonuclease. Step(c) and step (d) can be performed by adding the portion of the probenucleic acid comprising the initial amplifying restriction endonucleaseto a compartment comprising the first reporter nucleic acid and thesecond reporter nucleic acid. The probe nucleic acid can comprise (i) asingle-stranded portion comprising the nucleotide sequence complementaryto the sequence of the target nucleic acid and (ii) a double-strandedportion. The probe nucleic acid can comprise a first nucleic acidstrand, which can comprise the nucleotide sequence complementary to thesequence of the target nucleic acid, hybridized to a second nucleic acidstrand comprising the initial amplifying restriction endonuclease. Thefirst nucleic acid strand can be attached to a solid support. The firstnucleic acid strand can be directly attached to a solid support. Aportion of the second nucleic acid strand can be hybridized with thefirst nucleic acid strand to form the double-stranded portion. Theportion of the probe nucleic acid comprising the initial amplifyingrestriction endonuclease that is separated from the at least anotherportion of the probe nucleic acid in step (b) can comprise a portion ofthe first nucleic acid strand and all of the second strand. The portionof the probe nucleic acid comprising the initial amplifying restrictionendonuclease that is separated from the at least another portion of theprobe nucleic acid in step (b) can comprise at least a portion of thetarget nucleic acid. The method can comprise using a plurality of theprobe nucleic acid in step (a). The method can comprise using aplurality of the first reporter nucleic acid in step (c). The firstreporter nucleic acid in step (c) can be in molar excess of the portionof the probe nucleic acid comprising the initial amplifying restrictionendonuclease from step (b). The method can comprise using a plurality ofthe second reporter nucleic acid in step (d). The second reporternucleic acid in step (d) can be in molar excess of the portion of theprobe nucleic acid comprising the initial amplifying restrictionendonuclease from step (b). The number of molecules of the portion ofthe probe nucleic acid comprising the initial amplifying restrictionendonuclease that is separated from the at least another portion of theprobe nucleic acid in step (b) can be in an essentially linearrelationship to the number of molecules of the target nucleic acidpresent in the sample. The first reporter nucleic acid and the secondreporter nucleic acid can be attached to a solid support. The firstreporter nucleic acid and the second reporter nucleic acid can bedirectly attached to a solid support. The first reporter nucleic acidand the second reporter nucleic acid can be attached to a solid supportin the same compartment. The portion of the first reporter nucleic acidcomprising the secondary amplifying restriction endonuclease can bereleased from the solid support via step (c). The portion of the secondreporter nucleic acid comprising the initial amplifying restrictionendonuclease can be released from the solid support via step (d). Thefirst reporter nucleic acid can comprise a label. The label can be afluorescent label, a radioactive label, an enzyme label, or a redoxlabel. The second reporter nucleic acid can comprise a label. The labelcan be a fluorescent label, a radioactive label, an enzyme label, or aredox label. The first reporter nucleic acid and the second reporternucleic acid can comprise a label. The first reporter nucleic acid andthe second reporter nucleic acid can comprise the same label. The labelcan be a fluorescent label, a radioactive label, an enzyme label, or aredox label. The first reporter nucleic acid can be attached to a solidsupport, wherein the portion of the first reporter nucleic acid that isseparated from the at least another portion of the first reporternucleic acid comprises a label, and wherein the portion of the firstreporter nucleic acid that is separated from the at least anotherportion of the first reporter nucleic acid and that comprises the labelis released from the solid support via step (c). The first reporternucleic acid can comprise a first nucleic acid strand, which cancomprise the secondary amplifying restriction endonuclease, hybridizedto a second nucleic acid strand to form the double-stranded portion ofnucleic acid comprising the restriction endonuclease cut site of theinitial amplifying restriction endonuclease. The first nucleic acidstrand can be attached to a solid support. The first nucleic acid strandcan be directly attached to a solid support. The second nucleic acidstrand can be attached to a solid support. The second nucleic acidstrand can be directly attached to a solid support. The first nucleicacid strand can comprise a label. The label can be a fluorescent label,a radioactive label, an enzyme label, or a redox label. The secondnucleic acid strand can comprise a label. The label can be a fluorescentlabel, a radioactive label, an enzyme label, or a redox label. Thesecond reporter nucleic acid can be attached to a solid support, whereinthe portion of the second reporter nucleic acid that is separated fromthe at least another portion of the second reporter nucleic acidcomprises a label, and wherein the portion of the second reporternucleic acid that is separated from the at least another portion of thesecond reporter nucleic acid and that comprises the label is releasedfrom the solid support via step (d). The second reporter nucleic acidcan comprise a first nucleic acid strand, which can comprise the initialamplifying restriction endonuclease, hybridized to a second nucleic acidstrand to form the double-stranded portion of nucleic acid comprisingthe restriction endonuclease cut site of the secondary amplifyingrestriction endonuclease. The first nucleic acid strand can be attachedto a solid support. The first nucleic acid strand can be directlyattached to a solid support. The second nucleic acid strand can beattached to a solid support. The second nucleic acid strand can bedirectly attached to a solid support. The first nucleic acid strand cancomprise a label. The label can be a fluorescent label, a radioactivelabel, an enzyme label, or a redox label. The second nucleic acid strandcan comprise a label. The label can be a fluorescent label, aradioactive label, an enzyme label, or a redox label. The portion of thefirst reporter nucleic acid separated from the at least another portionof the first reporter nucleic acid can comprise a fluorescent label,wherein the portion of the second reporter nucleic acid separated fromthe at least another portion of the second reporter nucleic acidcomprises a fluorescent label, and wherein determining step (e)comprises detecting the fluorescent label. Determining step (e) cancomprise detecting the portion of the first reporter nucleic acidseparated from the at least another portion of the first reporternucleic acid using a capillary electrophoresis technique. Determiningstep (e) can comprise detecting the portion of the second reporternucleic acid separated from the at least another portion of the secondreporter nucleic acid using a capillary electrophoresis technique. Steps(a), (b), (c), and (d) can be performed without nucleic acidamplification. Steps (a), (b), (c), (d), and (e) can be performedwithout nucleic acid amplification. The determining step can comprisedetermining the amount of the target nucleic acid present within thesample.

In another aspect, this document features a kit for assessing a samplefor target nucleic acid. The kit comprises, or consists essentially of,a probe nucleic acid comprising an amplifying restriction endonucleaseand a nucleotide sequence complementary to a sequence of the targetnucleic acid, wherein at least a portion of the target nucleic acid iscapable of hybridizing to at least a portion of the probe nucleic acidto form a double-stranded portion of nucleic acid comprising arestriction endonuclease cut site. The probe nucleic acid can besingle-stranded probe nucleic acid. The kit can comprise a solidsupport, and the probe nucleic acid can be attached to the solidsupport. A portion of the probe nucleic acid comprising the amplifyingrestriction endonuclease can be releasable from the solid support viacleavage with a recognition restriction endonuclease having the abilityto cleave at the restriction endonuclease cut site. The kit can furthercomprise the recognition restriction endonuclease. The probe nucleicacid can comprise (i) a single-stranded portion comprising thenucleotide sequence complementary to the sequence of the target nucleicacid and (ii) a double-stranded portion. The probe nucleic acid cancomprise a first nucleic acid strand, which can comprise the nucleotidesequence complementary to the sequence of the target nucleic acid,hybridized to a second nucleic acid strand comprising the amplifyingrestriction endonuclease. The kit can further comprise a reporternucleic acid comprising a double-stranded portion of nucleic acidcomprising a restriction endonuclease cut site of the amplifyingrestriction endonuclease. The kit can comprise a solid support, and thereporter nucleic acid can be attached to the solid support. The reporternucleic acid can be directly attached to the solid support. The reporternucleic acid can comprise a single-stranded portion of nucleic acid. Thereporter nucleic acid can comprise a label. The label can be afluorescent label, a radioactive label, an enzyme label, or a redoxlabel. A portion of the reporter nucleic acid comprising the label canbe capable of being separated from at least another portion of thereporter nucleic acid via cleavage by the amplifying restrictionendonuclease. The reporter nucleic acid can comprise a first nucleicacid strand, which can comprise the label, hybridized to a secondnucleic acid strand. The kit can further comprise (a) a first signalexpansion nucleic acid comprising a secondary amplifying restrictionendonuclease and a double-stranded section having a restrictionendonuclease cut site for the amplifying restriction endonuclease, and(b) a second signal expansion nucleic acid comprising the amplifyingrestriction endonuclease and a double-stranded section having arestriction endonuclease cut site for the secondary amplifyingrestriction endonuclease.

In another aspect, this document features a composition comprising, orconsisting essentially of, nucleic acid covalently attached to arestriction endonuclease having the ability to cleave a double-strandedDNA molecule. The nucleic acid can comprise a single-stranded sectionhaving the ability to hybridize to target nucleic acid.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting an exemplary method for detecting targetnucleic acid using probe nucleic acid, a recognition restrictionendonuclease, and reporter nucleic acid.

FIG. 2 is a schematic of an exemplary configuration of probe nucleicacid that can be used with the methods and materials provided herein fordetecting target nucleic acid.

FIG. 3 is a schematic depicting an exemplary method for detecting targetnucleic acid using probe nucleic acid, a recognition restrictionendonuclease, first signal expansion nucleic acid, second signalexpansion nucleic acid, and reporter nucleic acid.

FIG. 4 is a schematic of an exemplary configuration of first signalexpansion nucleic acid and second signal expansion nucleic acid that canbe used with the methods and materials provided herein for detectingtarget nucleic acid. Such first signal expansion nucleic acid and secondsignal expansion nucleic acid can be used with or without reporternucleic acid. When used without a separate reporter nucleic acid step,such signal expansion nucleic acid can be referred to as reporternucleic acid.

FIG. 5 is a schematic of an exemplary configuration of first signalexpansion nucleic acid and second signal expansion nucleic acid that canbe used with the methods and materials provided herein for detectingtarget nucleic acid. Such first signal expansion nucleic acid and secondsignal expansion nucleic acid can be used with or without reporternucleic acid. When used without a separate reporter nucleic acid step,such signal expansion nucleic acid can be referred to as reporternucleic acid.

FIG. 6 contains line graphs demonstrating the effect of targetoligonucleotide concentration (A) and recognition restrictionendonuclease concentration (B) on the cleavage of HRP-labeled nucleicacid as detected by the formation of colored reaction product.

DETAILED DESCRIPTION

This document provides methods and materials for detecting targetnucleic acid. For example, this document provides methods and materialsfor detecting the presence or absence of target nucleic acid within asample, methods and materials for detecting the amount of target nucleicacid present within a sample, kits for detecting the presence or absenceof target nucleic acid within a sample, kits for detecting the amount oftarget nucleic acid present within a sample, and methods for making suchkits.

In one embodiment, a method for detecting target nucleic acid caninclude contacting a sample (e.g., a sample to be tested or suspected tocontain target nucleic acid) with probe nucleic acid. The probe nucleicacid can be designed to have a single-stranded portion with a nucleotidesequence that is complementary to at least a portion of the targetnucleic acid to be detected. In this case, target nucleic acid presentwithin the sample can hybridize with the complementary sequence of thissingle-stranded portion of the probe nucleic acid to form adouble-stranded section with one strand being target nucleic acid andthe other strand being probe nucleic acid. In addition, thesingle-stranded portion of the probe nucleic acid having the nucleotidesequence that is complementary to at least a portion of the targetnucleic acid to be detected can be designed such that hybridization withthe target nucleic acid creates a restriction endonuclease cut site.Thus, target nucleic acid present within the sample can hybridize withthe complementary sequence of the single-stranded portion of the probenucleic acid to form a double-stranded section that creates a cut sitefor a restriction endonuclease. This cut site created by thehybridization of target nucleic acid to probe nucleic acid can bereferred to as a recognition restriction endonuclease cut site. Inaddition, a restriction endonuclease that cleaves nucleic acid at such arecognition restriction endonuclease cut site can be referred to as arecognition restriction endonuclease.

The probe nucleic acid also can be designed to contain a restrictionendonuclease. This restriction endonuclease, which can be a component ofthe probe nucleic acid, can be referred to as an amplifying restrictionendonuclease. An amplifying restriction endonuclease is typically adifferent restriction endonuclease than the restriction endonucleasethat is used as a recognition restriction endonuclease. For example,when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., a Hind III restriction endonuclease) isused as an amplifying restriction endonuclease. Thus, in general, probenucleic acid is designed to contain an amplifying restrictionendonuclease and to have a nucleotide sequence such that the targetnucleic acid can hybridize to the probe nucleic acid and create arecognition restriction endonuclease cut site for a recognitionrestriction endonuclease. In some cases, the probe nucleic acid can beattached to a solid support (e.g., a well of a microtiter plate). Forexample, the probe nucleic acid can be attached to a solid support suchthat cleavage at the recognition restriction endonuclease cut site viathe recognition restriction endonuclease releases a portion of the probenucleic acid that contains the amplifying restriction endonuclease.

After contacting the sample that may or may not contain target nucleicacid with the probe nucleic acid that is attached to a solid support,the target nucleic acid, if present in the sample, can hybridize to theprobe nucleic acid and create the recognition restriction endonucleasecut site. At this point, the recognition restriction endonuclease,whether added to the reaction or already present in the reaction, cancleave the probe nucleic acid at the recognition restrictionendonuclease cut sites that are formed by the hybridization of targetnucleic acid to the probe nucleic acid, thereby releasing the portion ofthe probe nucleic acid that contains the amplifying restrictionendonuclease from the solid support. The number of amplifyingrestriction endonuclease-containing portions of the probe nucleic acidthat are released from the solid support can be in an essentially linearrelationship (e.g., essentially a one-for-one relationship) with thenumber of target nucleic acid molecules that hybridize with the probenucleic acid to form the recognition restriction endonuclease cut site.

The portions of the probe nucleic acid containing the amplifyingrestriction endonuclease that were released from the solid support canbe collected and placed in contact with reporter nucleic acid. Forexample, the released portions of the probe nucleic acid, if present,can be transferred from one well of a microtiter plate (e.g., a 96-wellplate) that contained the probe nucleic acid to another well of amicrotiter plate that contains the reporter nucleic acid. The reporternucleic acid can be designed to have a double-stranded portion with arestriction endonuclease cut site for the amplifying restrictionendonuclease of the probe nucleic acid. This restriction endonucleasecut site for the amplifying restriction endonuclease can be referred toas an amplifying restriction endonuclease cut site. If portions of theprobe nucleic acid containing the amplifying restriction endonucleaseare present and placed in contact with the reporter nucleic acid, thenthe reporter nucleic acid can be cleaved at the amplifying restrictionendonuclease cut site by the amplifying restriction endonuclease. Sincethe amplifying restriction endonucleases of the released portions of theprobe nucleic acid are free to carry out repeated cleavage events, thenumber of reporter nucleic acid molecules that are cleaved can greatlyexceed the number of amplifying restriction endonucleases present in thereaction. For example, the number of cleaved reporter nucleic acidmolecules can greatly exceed (e.g., exponentially exceed) the number ofamplifying restriction endonucleases present in the reaction andtherefore can greatly exceed (e.g., exponentially exceed) the number oftarget nucleic acid molecules that were present in the sample contactedwith the probe nucleic acid. Such a greatly expanded relationship (e.g.,an exponential relationship) can allow very small amounts of targetnucleic acid present in the sample to be readily detected.

After the released portions of the probe nucleic acid, if present, arecontacted with the reporter nucleic acid, the presence or absence ofcleaved reporter nucleic acid can be determined. The presence of cleavedreporter nucleic acid can indicate that the sample contained the targetnucleic acid, while the absence of cleaved reporter nucleic acid canindicate that the sample lacked the target nucleic acid. In some cases,the amount of cleaved reporter nucleic acid can be determined. In suchcases, the amount of cleaved reporter nucleic acid can indicate theamount of target nucleic acid present in the sample. A standard curveusing known amounts of target nucleic acid can be used to aid in thedetermination of the amount of target nucleic acid present within asample.

In some cases, the reporter nucleic acid can contain a label to aid inthe detection of cleaved reporter nucleic acid. For example, reporternucleic acid can contain a fluorescent label and a quencher such thatcleaved reporter nucleic acid provides a fluorescent signal anduncleaved reporter nucleic acid does not provide a fluorescent signal.In some cases, the reporter nucleic acid can contain a label (e.g., acolorimetric label, a fluorescent label or an enzyme such as horseradish peroxidase) and can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the reporter nucleic acid can beattached to a solid support such that cleavage at the amplifyingrestriction endonuclease cut site by the amplifying restrictionendonuclease releases a portion of the reporter nucleic acid thatcontains the label. The resulting reaction mixture can be collected andassessed for the presence, absence, or amount of released portions ofthe reporter nucleic acid using the label. For example, the releasedportions of the reporter nucleic acid, if present, can be transferredfrom one well of a microtiter plate (e.g., a 96-well plate) thatcontained the reporter nucleic acid to another well of a microtiterplate, where the transferred material can be assessed for a signal fromthe label.

One example of a method of detecting target nucleic acid that includesusing probe nucleic acid and reporter nucleic acid is set forth inFIG. 1. With reference to FIG. 1, first reaction chamber 100 (e.g., amicrotiter plate well) can contain probe nucleic acid 101. Probe nucleicacid 101 can be attached (e.g., immobilized) to solid support 102 andcan include amplifying restriction endonuclease 103 (Ra). Probe nucleicacid 101 can be attached to solid support 102 such that amplifyingrestriction endonuclease 103 is released from solid support 102 uponcleavage of a nucleic acid component of probe nucleic acid 101. Probenucleic acid 101 can have a single-stranded section having a nucleotidesequence that is complementary to at least a portion of target nucleicacid 104. Probe nucleic acid 101 can be contacted with a sample that mayor may not contain target nucleic acid 104. If target nucleic acid 104is present, at least a portion of target nucleic acid 104 and probenucleic acid 101 can hybridize to form a double-stranded section ofnucleic acid. Such a double-stranded section can contain at least onerecognition restriction endonuclease cut site 105. Addition ofrecognition restriction endonuclease 106 (Rr) to first reaction chamber100 can result in the cleave of probe nucleic acid 101 at recognitionrestriction endonuclease cut site 105 formed by one strand of probenucleic acid and one strand of target nucleic acid, thereby releasingportion 107 of probe nucleic acid 101 from solid support 102. Portion107 can include amplifying restriction endonuclease 103.

The reaction product from first reaction chamber 100 containing releasedportion 107, if target nucleic acid 104 was present, can be transferred(e.g., manually or automatically) to second reaction chamber 120. Secondreaction chamber 120 can contain reporter nucleic acid 121. Reporternucleic acid 121 can be attached (e.g., immobilized) to solid support122 and can include marker (e.g., a label) 123 (M). Reporter nucleicacid 121 can be attached to solid support 122 such that marker 123 isreleased from solid support 122 upon cleavage of a nucleic acidcomponent of reporter nucleic acid 121. Reporter nucleic acid 121 canhave at least one double-stranded portion that contains at least oneamplifying restriction endonuclease cut site 124. Addition of thereaction product from first reaction chamber 100 to second reactionchamber 120 can result in the cleavage of reporter nucleic acid 121 atamplifying restriction endonuclease cut site 124 if the reaction productcontains portion 107. Such cleavage of reporter nucleic acid 121 canresult in the release of portion 127 from solid support 122. Portion 127can include marker 123.

The reaction product from second reaction chamber 120 can be assessed todetermine the presence, absence, or amount of portion 127. The presenceof portion 127 can indicate that the sample contained target nucleicacid 104, while the absence of portion 127 can indicate that the samplelacked target nucleic acid 104. In some cases, the amount of portion 127can be determined. In such cases, the amount of portion 127 can indicatethe amount of target nucleic acid 104 present in the sample. Thepresence, absence, or amount of portion 127 can be determined usingmarker 123, and portion 127 having marker 123 can be distinguished fromuncleaved reporter nucleic acid 121 having marker 123 since, in thisexample, portion 127 is released from solid support 122, while uncleavedreporter nucleic acid 121 remains attached to solid support 122. Forexample, in some cases, the reaction product from second reactionchamber 120 can be transferred to third reaction chamber where thepresence or absence of portion 127 via marker 123 is assessed. Ifportion 127 is present, the amount of portion 127 present can bequantified.

Probe nucleic acid 101 and reporter nucleic acid 121 can have variousconfigurations. For example, with reference to FIG. 1, probe nucleicacid 101 can be designed to have a single nucleic acid strand such thatthe entire nucleic acid component of probe nucleic acid 101 issingle-stranded prior to contact with target nucleic acid 104. Inanother example, with reference to FIG. 2, probe nucleic acid 101 can bedesigned to have first strand 128 and second strand 108. First strand128 can be attached to solid support 102 and can be designed to have asingle-stranded section having a nucleotide sequence that iscomplementary to at least a portion of target nucleic acid 104. Secondstrand 108 can include amplifying restriction endonuclease 103 and canhave a single-stranded section having a nucleotide sequence that canhybridize to first strand 128. In some cases, first strand 128 andsecond strand 108 can be synthesized or obtained separately and thenmixed together to form probe nucleic acid 101. For example, first strand128 can be synthesized, biotinylated, and attached to astreptavidin-coated solid support. After synthesizing the nucleic acidcomponent of second strand 108 and attaching amplifying restrictionendonuclease 103 to the synthesized nucleic acid component, secondstrand 108 can be incubated with first strand 128 to form nucleic acidprobe 101. In some cases, probe nucleic acid 101 can contain more thantwo strands. For example, probe nucleic acid can include first strand150, second strand 152, and third strand 154. In this case, first strand150 can be attached to solid support 102, second strand 152 can behybridized to first strand 150 and can include a single-stranded sectionhaving a nucleotide sequence that is complementary to at least a portionof target nucleic acid 104, and third strand 154 can be hybridized tosecond strand 152 and can be attached to amplifying restrictionendonuclease 103. Similar one, two, three, or more strand configurationscan be used to make reporter nucleic acid.

In another embodiment, a method for detecting target nucleic acid caninclude contacting a sample (e.g., a sample to be tested or suspected tocontain target nucleic acid) with probe nucleic acid. The probe nucleicacid can be designed to have a single-stranded portion with a nucleotidesequence that is complementary to at least a portion of the targetnucleic acid to be detected. In this case, target nucleic acid presentwithin the sample can hybridize with the complementary sequence of thissingle-stranded portion of the probe nucleic acid to form adouble-stranded section with one strand being target nucleic acid andthe other strand being probe nucleic acid. In addition, thesingle-stranded portion of the probe nucleic acid having the nucleotidesequence that is complementary to at least a portion of the targetnucleic acid to be detected can be designed such that hybridization withthe target nucleic acid creates a recognition restriction endonucleasecut site. Thus, target nucleic acid present within the sample canhybridize with the complementary sequence of the single-stranded portionof the probe nucleic acid to form a double-stranded section that createsa recognition restriction endonuclease cut site for a recognitionrestriction endonuclease. The probe nucleic acid also can be designed tocontain an amplifying restriction endonuclease. Since this methodincludes the use of two or more different amplifying restrictionendonucleases, the amplifying restriction endonuclease that is acomponent of the probe nucleic acid can be referred to as a first or aninitial amplifying restriction endonuclease, with additional amplifyingrestriction endonucleases being referred to as second, third, and so onor secondary, tertiary, and so on amplifying restriction endonucleases.This initial amplifying restriction endonuclease is typically adifferent restriction endonuclease than the restriction endonucleasethat is used as a recognition restriction endonuclease. For example,when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., a Hind III restriction endonuclease) isused as an initial amplifying restriction endonuclease. Thus, ingeneral, probe nucleic acid is designed to contain an initial amplifyingrestriction endonuclease and to have a nucleotide sequence such that thetarget nucleic acid can hybridize to the probe nucleic acid and create arecognition restriction endonuclease cut site for a recognitionrestriction endonuclease. In some cases, the probe nucleic acid can beattached to a solid support (e.g., a well of a microtiter plate). Forexample, the probe nucleic acid can be attached to a solid support suchthat cleavage at the recognition restriction endonuclease cut site viathe recognition restriction endonuclease releases a portion of the probenucleic acid that contains the initial amplifying restrictionendonuclease.

After contacting the sample that may or may not contain target nucleicacid with the probe nucleic acid that is attached to a solid support,the target nucleic acid, if present in the sample, can hybridize to theprobe nucleic acid and create the recognition restriction endonucleasecut site. At this point, the recognition restriction endonuclease,whether added to the reaction or already present in the reaction, cancleave the probe nucleic acid at the recognition restrictionendonuclease cut sites that are formed by the hybridization of targetnucleic acid to the probe nucleic acid, thereby releasing the portion ofthe probe nucleic acid that contains the initial amplifying restrictionendonuclease from the solid support. The number of initial amplifyingrestriction endonuclease-containing portions of the probe nucleic acidthat are released from the solid support can be in an essentially linearrelationship (e.g., essentially a one-for-one relationship) with thenumber of target nucleic acid molecules that hybridize with the probenucleic acid to form the recognition restriction endonuclease cut site.

The portions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease that were released from the solid support canbe collected and placed in contact with first signal expansion nucleicacid and second signal expansion nucleic acid. The first signalexpansion nucleic acid can be designed to have a double-stranded portionwith a restriction endonuclease cut site for the initial amplifyingrestriction endonuclease of the probe nucleic acid. This restrictionendonuclease cut site for the initial amplifying restrictionendonuclease can be referred to as an initial amplifying restrictionendonuclease cut site. The first signal expansion nucleic acid also canbe designed to contain a secondary amplifying restriction endonuclease.The second signal expansion nucleic acid can be designed to have adouble-stranded portion with a restriction endonuclease cut site for thesecondary amplifying restriction endonuclease of the first signalexpansion nucleic acid. This restriction endonuclease cut site for thesecondary amplifying restriction endonuclease can be referred to as asecondary amplifying restriction endonuclease cut site. The secondsignal expansion nucleic acid also can be designed to contain an initialamplifying restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease and a HindIII restriction endonuclease is used as aninitial amplifying restriction endonuclease of the probe nucleic acid, aSmaI restriction endonuclease can be used as a secondary amplifyingrestriction endonuclease of the first signal expansion nucleic acid anda HindIII restriction endonuclease can be used as the initial amplifyingrestriction endonuclease of the second signal expansion nucleic acid.

In some cases, the first signal expansion nucleic acid and second signalexpansion nucleic acid can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the first signal expansion nucleicacid can be attached to a solid support such that cleavage at theinitial amplifying restriction endonuclease cut site via the initialamplifying restriction endonuclease releases a portion of the firstsignal expansion nucleic acid that contains the secondary amplifyingrestriction endonuclease, and the second signal expansion nucleic acidcan be attached to a solid support such that cleavage at the secondaryamplifying restriction endonuclease cut site via the secondaryamplifying restriction endonuclease releases a portion of the secondsignal expansion nucleic acid that contains the initial amplifyingrestriction endonuclease. The first signal expansion nucleic acid can beattached to the same solid support (e.g., two different sub-compartmentsof a larger compartment) that contains the second signal expansionnucleic acid provided that the secondary amplifying restrictionendonuclease of uncleaved first signal expansion nucleic acid is unableto cleave the second signal expansion nucleic acid and provided that theinitial amplifying restriction endonuclease of uncleaved second signalexpansion nucleic acid is unable to cleave the first signal expansionnucleic acid. In some cases, the first signal expansion nucleic acid canbe attached to the same solid support within a joint compartment suchthat the first signal expansion nucleic acid is within a firstcompartment of the joint compartment and the second signal expansionnucleic acid is within a second compartment of the joint compartment. Insuch cases, the secondary amplifying restriction endonuclease ofuncleaved first signal expansion nucleic acid in the first compartmentis unable to cleave the second signal expansion nucleic acid located inthe second compartment, while the secondary amplifying restrictionendonuclease of cleaved first signal expansion nucleic acid is capableof moving (e.g., diffusing) from the first compartment to the secondcompartment to cleave the second signal expansion nucleic acid locatedin the second compartment. In addition, the initial amplifyingrestriction endonuclease of uncleaved second signal expansion nucleicacid in the second compartment is unable to cleave the first signalexpansion nucleic acid located in the first compartment, while theinitial amplifying restriction endonuclease of cleaved second signalexpansion nucleic acid is capable of moving (e.g., diffusing) from thesecond compartment to the first compartment to cleave the first signalexpansion nucleic acid located in the first compartment.

If portions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease are present and placed in contact with thefirst signal expansion nucleic acid, then the first signal expansionnucleic acid can be cleaved at the initial amplifying restrictionendonuclease cut site by the initial amplifying restrictionendonuclease, thereby releasing a portion of the first signal expansionnucleic acid that contains the secondary amplifying restrictionendonuclease from the solid support. The released portions of the firstsignal expansion nucleic acid containing the secondary amplifyingrestriction endonuclease can be free to cleave the second signalexpansion nucleic acid at the secondary amplifying restrictionendonuclease cut site, thereby releasing a portion of the second signalexpansion nucleic acid that contains the initial amplifying restrictionendonuclease from the solid support. Since the initial amplifyingrestriction endonucleases of the released portions of the probe nucleicacid, the initial amplifying restriction endonucleases of the releasedportions of the second signal expansion nucleic acid, and the secondaryamplifying restriction endonucleases of the released portions of thefirst signal expansion nucleic acid are free to carry out repeatedcleavage events, the number of released portions containing the initialamplifying restriction endonucleases is greatly increased from thenumber that were released by the recognition restriction endonuclease.For example, the number of cleaved first signal expansion nucleic acidmolecules can greatly exceed (e.g., exponentially exceed) the number ofreleased portions of the probe nucleic acid, and the number of cleavedsecond signal expansion nucleic acid molecules can greatly exceed (e.g.,exponentially exceed) the number of released portions of the probenucleic acid. Such a greatly expanded relationship (e.g., an exponentialrelationship) can allow very small amounts of target nucleic acidpresent in the sample to be readily detected.

In some cases, this method can be performed with the first signalexpansion nucleic acid being attached to a solid support that isdifferent from the solid support that contains the second signalexpansion nucleic acid. For example, the first signal expansion nucleicacid can be attached to one well of a microtiter plate, while the secondsignal expansion nucleic acid can be attached to a different well of amicrotiter plate. In this case, the resulting reaction material from thewell with the first signal expansion nucleic acid can be collected andtransferred to the well containing the second signal expansion nucleicacid.

The portions of the second signal expansion nucleic acid containing theinitial amplifying restriction endonuclease that were released from thesolid support containing the second signal expansion nucleic acid alongwith any other released portions in this reaction (e.g., the releasedportions of the probe nucleic acid containing the initial amplifyingrestriction endonuclease and the released portions of the first signalexpansion nucleic acid containing the secondary amplifying restrictionendonuclease) can be collected and placed in contact with reporternucleic acid. For example, the released portions, if present, can betransferred from one well of a microtiter plate (e.g., a 96-well plate)that contained the second signal expansion nucleic acid to another wellof a microtiter plate that contains the reporter nucleic acid. Thereporter nucleic acid can be designed to have a double-stranded portionwith a restriction endonuclease cut site for the initial amplifyingrestriction endonuclease. If released portions containing the initialamplifying restriction endonuclease are present and placed in contactwith the reporter nucleic acid, then the reporter nucleic acid can becleaved at the initial amplifying restriction endonuclease cut site bythe initial amplifying restriction endonuclease. Since the initialamplifying restriction endonucleases of the released portions are freeto carry out repeated cleavage events, the number of reporter nucleicacid molecules that are cleaved can greatly exceed the number of initialamplifying restriction endonucleases present in the reaction. Forexample, the number of cleaved reporter nucleic acid molecules cangreatly exceed (e.g., exponentially exceed) the number of initialamplifying restriction endonucleases present in the reaction andtherefore can greatly exceed (e.g., exponentially exceed) the number oftarget nucleic acid molecules that were present in the sample contactedwith the probe nucleic acid. Such a greatly expanded relationship (e.g.,an exponential relationship) can allow very small amounts of targetnucleic acid present in the sample to be readily detected.

After the released portions containing the initial amplifyingrestriction endonuclease, if present, are contacted with the reporternucleic acid, the presence or absence of cleaved reporter nucleic acidcan be determined. The presence of cleaved reporter nucleic acid canindicate that the sample contained the target nucleic acid, while theabsence of cleaved reporter nucleic acid can indicate that the samplelacked the target nucleic acid. In some cases, the amount of cleavedreporter nucleic acid can be determined. In such cases, the amount ofcleaved reporter nucleic acid can indicate the amount of target nucleicacid present in the sample.

In some cases, the reporter nucleic acid can contain a label to aid inthe detection of cleaved reporter nucleic acid. For example, reporternucleic acid can contain a fluorescent label and a quencher such thatcleaved reporter nucleic acid provides a fluorescent signal anduncleaved reporter nucleic acid does not provide a fluorescent signal.In some cases, the reporter nucleic acid can contain a label (e.g., acolorimetric label, fluorescent label or an enzyme such as horse radishperoxidase) and can be attached to a solid support (e.g., a well of amicrotiter plate). For example, the reporter nucleic acid can beattached to a solid support such that cleavage at the initial amplifyingrestriction endonuclease cut site by the initial amplifying restrictionendonuclease releases a portion of the reporter nucleic acid thatcontains the label. The resulting reaction mixture can be collected andassessed for the presence, absence, or amount of released portions ofthe reporter nucleic acid using the label. For example, the releasedportions of the reporter nucleic acid, if present, can be transferredfrom one well of a microtiter plate (e.g., a 96-well plate) thatcontained the reporter nucleic acid to another well of a microtiterplate, where the transferred material can be assessed for a signal fromthe label.

In some cases, the presence or absence of cleaved first signal expansionnucleic acid, cleaved second signal expansion nucleic acid, or both canbe determined. The presence of such cleaved nucleic acid can indicatethat the sample contained the target nucleic acid, while the absence ofsuch cleaved nucleic acid can indicate that the sample lacked the targetnucleic acid. In some cases, the amount of cleaved first signalexpansion nucleic acid, cleaved second signal expansion nucleic acid, orboth can be determined. In such cases, the amount of cleaved nucleicacid can indicate the amount of target nucleic acid present in thesample. In these cases, the use of cleaved first signal expansionnucleic acid, cleaved second signal expansion nucleic acid, or both toassess the sample for target nucleic acid can be in addition to the useof a separate reporter nucleic acid step or can replace the use of aseparate reporter nucleic acid step. In some cases, the first signalexpansion nucleic acid, the second signal expansion nucleic acid, orboth can be labeled in a manner similar to that described herein for thereporter nucleic acid to aid in detection.

When the presence, absence, or amount of cleaved first signal expansionnucleic acid, cleaved second signal expansion nucleic acid, or both aredetermined to assess the sample for target nucleic acid, the firstsignal expansion nucleic acid can be referred to as a first reporternucleic acid and the second signal expansion nucleic acid can bereferred to as a second reporter nucleic acid even though they includeamplifying restriction endonucleases.

Examples of a method of detecting target nucleic acid that includesusing probe nucleic acid, first signal expansion nucleic acid, secondsignal expansion nucleic acid, and reporter nucleic acid are set forthin FIGS. 3-5. With reference to FIG. 3, first reaction chamber 200(e.g., a microtiter plate well) can contain probe nucleic acid 201.Probe nucleic acid 201 can be attached (e.g., immobilized) to solidsupport 202 and can include initial amplifying restriction endonuclease203 (Ra). Probe nucleic acid 201 can be attached to solid support 202such that initial amplifying restriction endonuclease 203 is releasedfrom solid support 202 upon cleavage of a nucleic acid component ofprobe nucleic acid 201. Probe nucleic acid 201 can have asingle-stranded section having a nucleotide sequence that iscomplementary to at least a portion of target nucleic acid 204. Probenucleic acid 201 can be contacted with a sample that may or may notcontain target nucleic acid 204. If target nucleic acid 204 is present,at least a portion of target nucleic acid 204 and probe nucleic acid 201can hybridize to form a double-stranded section of nucleic acid. Such adouble-stranded section can contain at least one recognition restrictionendonuclease cut site 205. Addition of recognition restrictionendonuclease 206 (Rr) to first reaction chamber 200 can result in thecleavage of probe nucleic acid 201 at recognition restrictionendonuclease cut site 205 formed by one strand of probe nucleic acid andone strand of target nucleic acid, thereby releasing portion 207 ofprobe nucleic acid 201 from solid support 202. Portion 207 can includeinitial amplifying restriction endonuclease 203.

The reaction product from first reaction chamber 200 containing releasedportion 207, if target nucleic acid 204 was present, can be transferred(e.g., manually or automatically) to second reaction chamber 220. Secondreaction chamber 220 can contain first signal expansion nucleic acid 226and second signal expansion nucleic acid 225. First signal expansionnucleic acid 226 can have at least one double-stranded portion thatcontains at least one initial amplifying restriction endonuclease cutsite 230. First signal expansion nucleic acid 226 can be attached (e.g.,immobilized) to solid support 222 and can include secondary amplifyingrestriction endonuclease 223 (Rb). First signal expansion nucleic acid226 can be attached to solid support 222 such that portion 234containing secondary amplifying restriction endonuclease 223 is releasedfrom solid support 222 upon cleavage of first signal expansion nucleicacid 226 at initial amplifying restriction endonuclease cut site 230.For clarity, frame E of FIG. 3 omits depicting one strand from thecleaved versions of first signal expansion nucleic acid 226 and secondsignal expansion nucleic acid 225.

Second signal expansion nucleic acid 225 can have at least onedouble-stranded portion that contains at least one secondary amplifyingrestriction endonuclease cut site 232. Second signal expansion nucleicacid 225 can be attached (e.g., immobilized) to solid support 222 andcan include initial amplifying restriction endonuclease 224. Secondsignal expansion nucleic acid 225 can be attached to solid support 222such that portion 236 containing initial amplifying restrictionendonuclease 224 is released from solid support 222 upon cleavage ofsecond signal expansion nucleic acid 225 at secondary amplifyingrestriction endonuclease cut site 232. Initial amplifying restrictionendonuclease 203 of probe nucleic acid 201 and initial amplifyingrestriction endonuclease 224 of second signal expansion nucleic acid 225can be the same restriction endonuclease. For example, both can be anEcoRI restriction endonuclease.

Addition of the reaction product from first reaction chamber 200 tosecond reaction chamber 220 can result in the cleavage of first signalexpansion nucleic acid 226 at initial amplifying restrictionendonuclease cut site 230 if the reaction product contains portion 207.Such cleavage of first signal expansion nucleic acid 226 can result inthe release of portion 234 from solid support 222. Portion 234, whichcan include secondary amplifying restriction endonuclease 223, canresult in the cleavage of second signal expansion nucleic acid 225 atsecondary amplifying restriction endonuclease cut site 232. Suchcleavage of second signal expansion nucleic acid 225 can result in therelease of portion 236 from solid support 222. Thus, this reaction canresult in the accumulation of released portions 234 and 236.

The reaction product from second reaction chamber 220 containingreleased portion 207, released portion 234, and released portion 236, iftarget nucleic acid 204 was present, can be transferred (e.g., manuallyor automatically) to third reaction chamber 240. Third reaction chamber240 can contain reporter nucleic acid 241. Reporter nucleic acid 241 canbe attached (e.g., immobilized) to solid support 242 and can includemarker (e.g., a label) 243 (M). Reporter nucleic acid 241 can beattached to solid support 242 such that marker 243 is released fromsolid support 242 upon cleavage of a nucleic acid component of reporternucleic acid 241. Reporter nucleic acid 241 can have at least onedouble-stranded portion that contains at least one initial amplifyingrestriction endonuclease cut site 246. Addition of the reaction productfrom second reaction chamber 220 to third reaction chamber 240 canresult in the cleavage of reporter nucleic acid 241 at initialamplifying restriction endonuclease cut site 246 if the reaction productcontains portion 207 and portion 236. In some cases, reporter nucleicacid 241 can include at least one double-stranded portion that containsat least one cut site for secondary amplifying restriction endonuclease223. In such cases, addition of the reaction product from secondreaction chamber 220 to third reaction chamber 240 can result in thecleavage of reporter nucleic acid 241 at the cut site for secondaryamplifying restriction endonuclease 223 if the reaction product containsportion 234. Cleavage of reporter nucleic acid 241 can result in therelease of portion 247 from solid support 242. Portion 247 can includemarker 243.

The reaction product from third reaction chamber 240 can be assessed todetermine the presence, absence, or amount of portion 247. The presenceof portion 247 can indicate that the sample contained target nucleicacid 204, while the absence of portion 247 can indicate that the samplelacked target nucleic acid 204. In some cases, the amount of portion 247can be determined. In such cases, the amount of portion 247 can indicatethe amount of target nucleic acid 204 present in the sample. Thepresence, absence, or amount of portion 247 can be determined usingmarker 243, and portion 247 having marker 243 can be distinguished fromuncleaved reporter nucleic acid 241 having marker 243 since, in thisexample, portion 247 is released from solid support 242, while uncleavedreporter nucleic acid 241 remains attached to solid support 242. Forexample, in some cases, the reaction product from third reaction chamber24 can be transferred to fourth reaction chamber where the presence orabsence of portion 247 via marker 243 is assessed. If portion 347 ispresent, the amount of portion 247 present can be quantified.

In some cases and with reference to FIGS. 4 and 5, first signalexpansion nucleic acid 226 can include marker (e.g., a label) 243 (M)and second signal expansion nucleic acid 225 can include marker (e.g., alabel) 243 (M). In such cases, cleavage of first signal expansionnucleic acid 226 and cleavage of second signal expansion nucleic acid225 can be assessed using marker 243 to determine the presence, absence,or amount of target nucleic acid within a sample. For example, detector250 can be used to detect marker 243 released from solid support 222.

Probe nucleic acid 201, first signal expansion nucleic acid 226, secondsignal expansion nucleic acid 225, and reporter nucleic acid 241 canhave various configurations. For example, with reference to FIG. 3,probe nucleic acid 201 can be designed to have a single nucleic acidstrand such that the entire nucleic acid component of probe nucleic acid201 is single-stranded prior to contact with target nucleic acid 204. Inanother example, probe nucleic acid 201 can be designed in a manner likeprobe nucleic acid 101 to have two or more strands. See, e.g., FIG. 2.For example, probe nucleic acid 201 can have a first strand and a secondstrand. The first strand can be attached to a solid support and can bedesigned to have a single-stranded section having a nucleotide sequencethat is complementary to at least a portion of target nucleic acid. Thesecond strand can include an initial amplifying restriction endonucleaseand can have a single-stranded section having a nucleotide sequence thatcan hybridize to the first strand. In some cases, the first strand andsecond strand can be synthesized or obtained separately and then mixedtogether to form probe nucleic acid 201. For example, the first strandcan be synthesized, biotinylated, and attached to a streptavidin-coatedsolid support. After synthesizing the nucleic acid component of thesecond strand and attaching an initial amplifying restrictionendonuclease to the synthesized nucleic acid component, the secondstrand can be incubated with the first strand to form nucleic acid probe201. In some cases, probe nucleic acid 201 can contain more than twostrands. For example, probe nucleic acid can include a first strand, asecond strand, and a third strand. In this case, the first strand can beattached to a solid support, the second strand can be hybridized to thefirst strand and can include a single-stranded section having anucleotide sequence that is complementary to at least a portion oftarget nucleic acid, and the third strand can be hybridized to thesecond strand and can be attached to an initial amplifying restrictionendonuclease. Similar one, two, three, or more strand configurations canbe used to make first signal expansion nucleic acid, second signalexpansion nucleic acid, or reporter nucleic acid. For example, firstsignal expansion nucleic acid and second signal expansion nucleic acidcan be designed to have a configuration as shown in FIG. 4 or 5.

Probe nucleic acid described herein typically includes at least onesingle-stranded DNA section that is designed to hybridize with a desiredtarget nucleic acid and thereby create a recognition restrictionendonuclease cut site. The other portions of the probe nucleic acid caninclude DNA, RNA, or other molecules. For example, probe nucleic acidcan include biotin such that the probe nucleic acid can be attached to astreptavidin-coated solid support. In some cases, the single-strandedsection of the probe nucleic acid that is designed to hybridize with adesired target nucleic acid and create a recognition restrictionendonuclease cut site can be RNA or a nucleic acid analog (e.g., apeptide nucleic acid (PNA)) provided that such a single-stranded sectioncan (i) hybridize with the desired target nucleic acid and (ii) create arecognition restriction endonuclease cut site with the complementarytarget nucleic acid sequence that is capable of being cleaved by therecognition restriction endonuclease. Examples of restrictionendonucleases that can be used as recognition restriction endonucleasesto cleave a recognition restriction endonuclease cut site that iscreated between an RNA section of the probe nucleic acid and a DNAsection of the target nucleic acid include, without limitation, HhaI,AluI, TaqI, HaeIII, EcoRI, HindIII, SalI, and MspI restrictionendonucleases.

Probe nucleic acid described herein can be any length provided that thesingle-stranded section of the probe nucleic acid that is designed tohybridize with a desired target nucleic acid is capable of hybridizingto the target nucleic acid and provided that the amplifying restrictionendonuclease of the probe nucleic acid is capable of cleaving itsamplifying restriction endonuclease cut site after the probe nucleicacid is cleaved by a recognition restriction endonuclease. In general,the single-stranded section of the probe nucleic acid that is designedto hybridize with a desired target nucleic acid can be between about 10and about 500 or more nucleotides (e.g., between about 10 and about 400nucleotides, between about 10 and about 300 nucleotides, between about10 and about 200 nucleotides, between about 10 and about 100nucleotides, between about 10 and about 50 nucleotides, between about 10and about 25 nucleotides, between about 20 and about 500 nucleotides,between about 30 and about 500 nucleotides, between about 40 and about500 nucleotides, between about 50 and about 500 nucleotides, betweenabout 15 and about 50 nucleotides, between about 15 and about 25nucleotides, between about 20 and about 50 nucleotides, or between about18 and about 25 nucleotides) in length. The recognition restrictionendonuclease cut site that will be created by the hybridization oftarget nucleic acid to this single-stranded section of the probe nucleicacid can be located at any position alone the single-stranded section.For example, the recognition restriction endonuclease cut site to becreated can be towards the 5′ end, towards the '3 end, or near thecenter of the single-stranded section of the probe nucleic acid. Ingeneral, the overall length of the probe nucleic acid described hereincan be between about 10 and about 2500 or more nucleotides (e.g.,between about 10 and about 2000 nucleotides, between about 10 and about1000 nucleotides, between about 10 and about 500 nucleotides, betweenabout 10 and about 400 nucleotides, between about 10 and about 300nucleotides, between about 10 and about 200 nucleotides, between about10 and about 100 nucleotides, between about 10 and about 50 nucleotides,between about 10 and about 25 nucleotides, between about 20 and about500 nucleotides, between about 30 and about 500 nucleotides, betweenabout 40 and about 500 nucleotides, between about 50 and about 500nucleotides, between about 75 and about 500 nucleotides, between about100 and about 500 nucleotides, between about 150 and about 500nucleotides, between about 15 and about 50 nucleotides, between about 15and about 25 nucleotides, between about 20 and about 50 nucleotides, orbetween about 18 and about 25 nucleotides) in length.

The recognition restriction endonuclease cut site to be created byhybridization of target nucleic acid to the probe nucleic acid can be acut site of any type of restriction endonuclease. In addition, any typeof restriction endonuclease can be used as a recognition restrictionendonuclease to cleave probe nucleic acid upon target nucleic acidhybridization. Examples of restriction endonucleases that can be used asrecognition restriction endonucleases include, without limitation,EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI,HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI,StuI, XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV,RsaI, TaiI, AasI, BbsI, BseLI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI,RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI,BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII,BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI,CspI, Esp3I, KspAI, NsiI, SapI, and TauI restriction endonucleases. Insome cases, nucleic acid encoding a naturally-occurring restrictionendonuclease can be genetically engineered to create a modifiedrestriction endonuclease that has the ability to recognize a particularcut site. Common computer algorithms can be used to locate restrictionendonuclease cut sites along the nucleotide sequence of any desiredtarget nucleic acid. Once located, the sequence of the restrictionendonuclease cut site along with additional flanking sequence (e.g., 5′flanking sequence, 3′ flanking sequence, or both 5′ and 3′ flankingsequence) can be used to design the complementary sequence of the probenucleic acid that is used to hybridize to the target nucleic acid andcreate the recognition restriction endonuclease cut site upon targetnucleic acid hybridization.

In general, probe nucleic acid can be designed to have a single-strandedsection that is designed to hybridize with desired target nucleic acidand to form a single recognition restriction endonuclease cut site upontarget nucleic acid hybridization. In some cases, probe nucleic acid canbe designed to have a single-stranded section that is designed tohybridize with desired target nucleic acid and to form more than one(e.g., two, three, four, five, six, seven, eight, nine, ten, or more)recognition restriction endonuclease cut site upon target nucleic acidhybridization. When more than one recognition restriction endonucleasecut site is used, the multiple recognition restriction endonuclease cutsites can be cut sites for the same restriction endonuclease or cutsites for different restriction endonucleases. For example, probenucleic acid can be designed to have a single-stranded section that isdesigned to hybridize with desired target nucleic acid and to form onerecognition restriction endonuclease cut site for an EcoRI recognitionrestriction endonuclease and one recognition restriction endonucleasecut site for an XbaI recognition restriction endonuclease upon targetnucleic acid hybridization. In such cases, each recognition restrictionendonuclease can be used individually or in combination (e.g., as amixture) to cleave probe nucleic acid that hybridized to target nucleicacid and formed the corresponding recognition restriction endonucleasecut site via such hybridization.

Probe nucleic acid can be designed such that any target nucleic acid canbe detected. Examples of target nucleic acid that can be detected usingthe methods and materials provided herein include, without limitation,human nucleic acid, microbial nucleic acid (e.g., bacterial, fungal, orprotozoan nucleic acid), viral nucleic acid, nucleic acid containing apoint mutation, SNP, or gene rearrangement, mammalian nucleic acid,methylated nucleic acid, and mRNA. When detecting RNA target nucleicacid, restriction endonucleases having the ability to cleave arecognition restriction endonuclease cut site that is created between aDNA section of the probe nucleic acid and the RNA target nucleic acidcan be used as recognition restriction endonucleases. Examples of suchrestriction endonucleases include, without limitation, HhaI, AluI, TaqI,HaeIII, EcoRI, HindIII, SalI, and MspI restriction endonucleases. Whendetecting methylated target nucleic acid (e.g., a methylated targetnucleic acid associated with cancer), restriction endonucleases havingthe ability to cleave a recognition restriction endonuclease cut sitethat includes a methylated nucleotide to be assessed can be used asrecognition restriction endonucleases. Examples of restrictionendonucleases having the ability to recognize methylated nucleotidesinclude, without limitation, DpnI, GlaI, HpaII, MspI, AciI, HhaI, andSssI restriction endonucleases. In such cases, a control can includedetecting the same target nucleic acid without the methylatednucleotide. In some cases, a combination of methylation insensitive andmethylation sensitive restriction endonucleases can be used to assess asample for methylated target nucleic acid. For example, similargeneration of cleavage products using both methylation insensitive andmethylation sensitive restriction endonucleases designed for the samesite can indicate that the target nucleic acid lacks methylation at thatsite, while an increased level of cleavage products using a methylationinsensitive restriction endonuclease as compared to the level generatedusing a methylation sensitive restriction endonuclease designed for thesame site can indicate that the target nucleic acid is methylated atthat site.

The nucleotide sequence of target nucleic acid to be detected can beobtained from, for example, common nucleic acid databases such asGenBank®. A portion of target nucleic acid sequence can be selectedusing a computer-based program. For example, a computer-based programcan be used to detect restriction endonuclease cut sites within aportion of target nucleic acid. Such information can be used to designprobe nucleic acid such that the single-stranded section creates atleast one recognition restriction endonuclease cut site uponhybridization of the target nucleic acid.

Any appropriate method can be used to obtain the nucleic acid componentof the probe nucleic acid. For example, common molecular cloning andchemical nucleic acid synthesis techniques can be used to obtain thenucleic acid component of the probe nucleic acid. In some cases, thenucleic acid component of the probe nucleic acid can be synthesizedusing commercially available automated oligonucleotide synthesizers suchas those available from Applied Biosystems (Foster City, Calif.). Insome cases, probe nucleic acids can be synthesized de novo using any ofa number of procedures widely available in the art. Examples of suchmethods of synthesis include, without limitation, the 0-cyanoethylphosphoramidite method (Beaucage et al., Tet. Let., 22:1859-1862 (1981))and the nucleoside H-phosphonate method (Garegg et al., Tet. Let.,27:4051-4054 (1986); Froehler et al., Nucl. Acid Res., 14:5399-5407(1986); Garegg et al., Tet. Let., 27:4055-4058 (1986); and Gaffney etal., Tet. Let., 29:2619-2622 (1988)). These methods can be performed bya variety of commercially-available automated oligonucleotidesynthesizers. In some cases, recombinant nucleic acid techniques such asPCR and those that include using restriction enzyme digestion andligation of existing nucleic acid sequences (e.g., genomic DNA or cDNA)can be used to obtain the nucleic acid component of the probe nucleicacid.

Probe nucleic acid described herein can be attached to a solid support.Examples of solid supports include, without limitation, a well of amicrotiter plate (e.g., a 96-well microtiter plate or ELISA plate),beads (e.g., magnetic, glass, plastic, or gold-coated beads), slides(e.g., glass or gold-coated slides), micro- or nano-particles (e.g.,carbon nanotubes), platinum solid supports, palladium solid supports,and a surface of a chamber or channel within a microfluidic device. Insome cases, a solid support can be a silicon oxide-based solid support,a plastic polymer-based solid support (e.g., a nylon, nitrocellulose, orpolyvinylidene fluoride-based solid support), or a biopolymer-based(e.g., a cross-linked dextran or cellulose-based solid support) solidsupport. Probe nucleic acid can be directly or indirectly attached to asolid support. For example, biotin can be a component of the probenucleic acid, and the probe nucleic acid containing biotin can beindirectly attached to a solid support that is coated with streptavidinvia a biotin-streptavidin interaction. In some cases, probe nucleic acidcan be attached to a solid support via a covalent or non-covalentinteraction. For example, probe nucleic acid can be covalently attachedto magnetic beads as described elsewhere (Albretsen et al., Anal.Biochem., 189(1):40-50 (1990)).

Probe nucleic acid can be designed to contain any type of restrictionendonuclease as an amplifying restriction endonuclease. In general, anamplifying restriction endonuclease of the probe nucleic acid istypically a different restriction endonuclease than the restrictionendonuclease that is used as a recognition restriction endonuclease. Forexample, when an EcoRI restriction endonuclease is used as a recognitionrestriction endonuclease, a restriction endonuclease other than an EcoRIrestriction endonuclease (e.g., a HindIII restriction endonuclease) isused as an amplifying restriction endonuclease. Examples of restrictionendonucleases that can be used as amplifying restriction endonucleasesinclude, without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI,PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI,CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseJI, BspTI, ClaI,EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI,KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI,BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauIrestriction endonucleases. Any number of molecules of the sameamplifying restriction endonuclease can be attached to one probe nucleicacid molecule. For example, a single probe nucleic acid molecule cancontain one, two, three, four, five, or more EcoRI amplifyingrestriction endonuclease molecules. In some cases, a single probenucleic acid molecule can contain two or more (e.g., two, three, four,five, or more) different types of amplifying restriction endonucleases.For example, a single probe nucleic acid molecule can contain threeEcoRI amplifying restriction endonuclease molecules and two BanIIamplifying restriction endonuclease molecules.

Any appropriate method can be used to attach an amplifying restrictionendonuclease to a nucleic acid component of the probe nucleic acid. Insome cases, an amplifying restriction endonuclease can be attached by anionic or covalent attachment. For example, covalent bonds such as amidebonds, disulfide bonds, and thioether bonds, or bonds formed bycrosslinking agents can be used. In some cases, a non-covalent linkagecan be used. The attachment can be a direct attachment or an indirectattachment. For example, a linker can be used to attach an amplifyingrestriction endonuclease to a nucleic acid component of the probenucleic acid. In some cases, nucleic acid can include a thiolmodification, and a restriction endonuclease can be conjugated to thethiol-containing nucleic acid based on succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) using techniquessimilar to those described elsewhere (Dill et al., Biosensors andBioelectronics, 20:736-742 (2004)). In some cases, a biotinylatednucleic acid and a streptavidin-containing restriction endonuclease canbe attached to one another via a biotin-streptavidin interaction. Arestriction endonuclease can be conjugated with streptavidin using, forexample, sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate. An amplifyingrestriction endonuclease can be attached at any location of a nucleicacid component of the probe nucleic acid. For example, an amplifyingrestriction endonuclease can be attached at an end (e.g., a 5′ end or 3′end) of a nucleic acid component, in the middle of a nucleic acidcomponent, or at any position along the length of a nucleic acidcomponent.

Signal expansion nucleic acid (e.g., first signal expansion nucleic acidand second signal expansion nucleic acid) and reporter nucleic aciddescribed herein typically include at least one double-stranded DNAsection that includes an amplifying restriction endonuclease cut site(e.g., an initial amplifying restriction endonuclease cut site, asecondary amplifying restriction endonuclease cut site, or a tertiaryamplifying restriction endonuclease cut site). The other portions of thesignal expansion nucleic acid or reporter nucleic acid can include DNA,RNA, or other molecules. For example, reporter nucleic acid can includebiotin such that the reporter nucleic acid can be attached to astreptavidin-coated solid support. In some cases, one or both strands ofthe double-stranded section of the signal expansion nucleic acid or thereporter nucleic acid that contains an amplifying restrictionendonuclease cut site can be RNA or a nucleic acid analog (e.g., apeptide nucleic acid (PNA)) provided that such a double-stranded sectionis capable of being cleaved by the amplifying restriction endonuclease.Examples of restriction endonucleases that can be used as amplifyingrestriction endonucleases to cleave a DNA:RNA hybrid section of signalexpansion nucleic acid or reporter nucleic acid include, withoutlimitation, HhaI, AluI, TaqI, HaeIII, EcoRI, HindIII, SalI, and MspIrestriction endonucleases.

Signal expansion nucleic acid or reporter nucleic acid described hereincan be any length provided that the double-stranded section thatcontains the amplifying restriction endonuclease cut site is capable ofbeing cleaved by the amplifying restriction endonuclease. In general,the double-stranded section of signal expansion nucleic acid or reporternucleic acid can be between about 10 and about 500 or more nucleotides(e.g., between about 10 and about 400 nucleotides, between about 10 andabout 300 nucleotides, between about 10 and about 200 nucleotides,between about 10 and about 100 nucleotides, between about 10 and about50 nucleotides, between about 10 and about 25 nucleotides, between about20 and about 500 nucleotides, between about 30 and about 500nucleotides, between about 40 and about 500 nucleotides, between about50 and about 500 nucleotides, between about 15 and about 50 nucleotides,between about 15 and about 25 nucleotides, between about 20 and about 50nucleotides, or between about 18 and about 25 nucleotides) in length. Insome cases, the double-stranded section of signal expansion nucleic acidor reporter nucleic acid can be between 5 and 50 nucleotides in length.The amplifying restriction endonuclease cut site of the signal expansionnucleic acid or the reporter nucleic acid can be located at any positionalone the double-stranded section. For example, the amplifyingrestriction endonuclease cut site can be towards the 5′ end, towards the'3 end, or near the center of the double-stranded section of the signalexpansion nucleic acid or the reporter nucleic acid. In general, theoverall length of signal expansion nucleic acid or reporter nucleic aciddescribed herein can be between about 10 and about 2500 or morenucleotides (e.g., between about 10 and about 2000 nucleotides, betweenabout 10 and about 1000 nucleotides, between about 10 and about 500nucleotides, between about 10 and about 400 nucleotides, between about10 and about 300 nucleotides, between about 10 and about 200nucleotides, between about 10 and about 100 nucleotides, between about10 and about 50 nucleotides, between about 10 and about 25 nucleotides,between about 20 and about 500 nucleotides, between about 30 and about500 nucleotides, between about 40 and about 500 nucleotides, betweenabout 50 and about 500 nucleotides, between about 75 and about 500nucleotides, between about 100 and about 500 nucleotides, between about150 and about 500 nucleotides, between about 15 and about 50nucleotides, between about 15 and about 25 nucleotides, between about 20and about 50 nucleotides, or between about 18 and about 25 nucleotides)in length.

The amplifying restriction endonuclease cut site of signal expansionnucleic acid or reporter nucleic acid described herein can be a cut siteof any type of restriction endonuclease. In addition, any type ofrestriction endonuclease can be used as an amplifying restrictionendonuclease to cleave signal expansion nucleic acid or reporter nucleicacid. Examples of restriction endonucleases that can be used asamplifying restriction endonucleases include, without limitation, EcoRI,EcoRII, BamHI, HindIII, TaqI, NotI, HinfI, Sau3A, PovII, SmaI, HaeIII,HgaI, AluI, EcoRV, EcoP15I, KpnI, PstI, SacI, SalI, ScaI, SphI, StuI,XbaI, AarI, BanII, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlaIV, RsaI,TaiI, AasI, BbsI, BseLI, BspTI, ClaI, EcoO109I, I-PpoI, NmuCI, RsrII,TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI, KasI, Acc65I, BbvCI, BseMI,BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI, AccB7I, BbvI, BseMII, BsrFI,Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI, BceAI, BseNI, BsrGI, CspI,Esp3I, KspAI, NsiI, SapI, and TauI restriction endonucleases.

In general, signal expansion nucleic acid or reporter nucleic acid canbe designed to have a double-stranded section that contains a singleamplifying restriction endonuclease cut site. In some cases, signalexpansion nucleic acid or reporter nucleic acid provided herein can bedesigned to have a double-stranded section that contains more than one(e.g., two, three, four, five, six, seven, eight, nine, ten, or more)amplifying restriction endonuclease cut site. When more than oneamplifying restriction endonuclease cut site is used, the multipleamplifying restriction endonuclease cut sites can be cut sites for thesame restriction endonuclease or cut sites for different restrictionendonucleases. For example, reporter nucleic acid can be designed tohave a double-stranded section that contains one initial amplifyingrestriction endonuclease cut site for an EcoRI initial amplifyingrestriction endonuclease and one secondary amplifying restrictionendonuclease cut site for an XbaI secondary amplifying restrictionendonuclease.

Any appropriate method can be used to obtain the nucleic acid componentof signal expansion nucleic acid or reporter nucleic acid. For example,common molecular cloning and chemical nucleic acid synthesis techniquescan be used to obtain the nucleic acid component of signal expansionnucleic acid or reporter nucleic acid. In some cases, the nucleic acidcomponent of signal expansion nucleic acid or reporter nucleic acid canbe synthesized using commercially available automated oligonucleotidesynthesizers such as those available from Applied Biosystems (FosterCity, Calif.). In some cases, signal expansion nucleic acid or reporternucleic acid can be synthesized de novo using any of a number ofprocedures widely available in the art. Examples of such methods ofsynthesis include, without limitation, the β-cyanoethyl phosphoramiditemethod (Beaucage et al., Tet. Let., 22:1859-1862 (1981)) and thenucleoside H-phosphonate method (Garegg et al., Tet. Let., 27:4051-4054(1986); Froehler et al., Nucl. Acid Res., 14:5399-5407 (1986); Garegg etal., Tet. Let., 27:4055-4058 (1986); and Gaffney et al., Tet. Let.,29:2619-2622 (1988)). These methods can be performed by a variety ofcommercially-available automated oligonucleotide synthesizers. In somecases, recombinant nucleic acid techniques such as PCR and those thatinclude using restriction enzyme digestion and ligation of existingnucleic acid sequences (e.g., genomic DNA or cDNA) can be used to obtainthe nucleic acid component of signal expansion nucleic acid or reporternucleic acid.

Signal expansion nucleic acid or reporter nucleic acid described hereincan be attached to a solid support. Examples of solid supports include,without limitation, a well of a microtiter plate (e.g., a 96-wellmicrotiter plate or ELISA plate), beads (e.g., magnetic, glass, plastic,or gold-coated beads), slides (e.g., glass or gold-coated slides),micro- or nano-particles (e.g., carbon nanotubes), platinum solidsupports, palladium solid supports, and a surface of a chamber orchannel within a microfluidic device. In some cases, a solid support canbe a silicon oxide-based solid support, a plastic polymer-based solidsupport (e.g., a nylon, nitrocellulose, or polyvinylidene fluoride-basedsolid support) or a biopolymer-based (e.g., a cross-linked dextran orcellulose-based solid support) solid support.

Signal expansion nucleic acid or reporter nucleic acid can be directlyor indirectly attached to a solid support. For example, biotin can be acomponent of signal expansion nucleic acid or reporter nucleic acid, andthe signal expansion nucleic acid or the reporter nucleic acidcontaining biotin can be indirectly attached to a solid support that iscoated with streptavidin via a biotin-streptavidin interaction. In somecases, signal expansion nucleic acid or reporter nucleic acid can beattached to a solid support via a covalent or non-covalent interaction.For example, signal expansion nucleic acid or reporter nucleic acid canbe covalently attached to magnetic beads as described elsewhere(Albretsen et al., Anal. Biochem., 189(1):40-50 (1990)).

Signal expansion nucleic acid can be designed to contain any type ofrestriction endonuclease as an amplifying restriction endonuclease(e.g., an initial amplifying restriction endonuclease, a secondaryamplifying restriction endonuclease, or a tertiary amplifyingrestriction endonuclease). In general, an amplifying restrictionendonuclease of signal expansion nucleic acid is typically a differentrestriction endonuclease than the restriction endonuclease that is usedas a recognition restriction endonuclease. For example, when an EcoRIrestriction endonuclease is used as a recognition restrictionendonuclease, a restriction endonuclease other than an EcoRI restrictionendonuclease (e.g., a HeaIII restriction endonuclease) is used as anamplifying restriction endonuclease. Examples of restrictionendonucleases that can be used as amplifying restriction endonucleasesinclude, without limitation, EcoRI, EcoRII, BamHI, HindIII, TaqI, NotI,HinfI, Sau3A, PovII, SmaI, HaeIII, HgaI, AluI, EcoRV, EcoP15I, KpnI,PstI, SacI, SalI, ScaI, SphI, StuI, XbaI, AarI, BanII, BseGI, BspPI,CfrI, EcoNI, Hsp92II, NlaIV, RsaI, TaiI, AasI, BbsI, BseJI, BspTI, ClaI,EcoO109I, I-PpoI, NmuCI, RsrII, TaqaI, AatII, BbuI, BseLI, BsrBI, CpoI,KasI, Acc65I, BbvCI, BseMI, BsrDI, Csp45I, Kpn2I, NruI, SacII, TasI,AccB7I, BbvI, BseMII, BsrFI, Csp6I, EheI, KpnI, NsbI, SalI, TatI, AccI,BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, NsiI, SapI, and TauIrestriction endonucleases. Any number of molecules of the sameamplifying restriction endonuclease can be attached to one signalexpansion nucleic acid molecule. For example, a single signal expansionnucleic acid molecule can contain one, two, three, four, five, or moreEcoRI amplifying restriction endonuclease molecules. In some cases, asingle signal expansion nucleic acid molecule can contain two or more(e.g., two, three, four, five, or more) different types of amplifyingrestriction endonucleases. For example, a single signal expansionnucleic acid molecule can contain three BanII amplifying restrictionendonuclease molecules and two SacII amplifying restriction endonucleasemolecules.

Reporter nucleic acid can be designed to contain a label to aid in thedetection of cleaved reporter nucleic acid. In some cases, signalexpansion nucleic acid can be designed to contain a label. In suchcases, signal expansion nucleic acid containing a label can be used inaddition to reporter nucleic acid or in place of reporter nucleic acidto detect target nucleic acid. Examples of labels that can be acomponent of reporter nucleic acid or signal expansion nucleic acidinclude, without limitation, fluorescent labels (with or without the useof quenchers), dyes, antibodies, radioactive material, enzymes (e.g.,horse radish peroxidase, alkaline phosphatese, laccase, galactosidase,or luciferase), redox labels (e.g., ferrocene redox labels), metallicparticles (e.g., gold nanoparticles), green fluorescent protein-basedlabels. In some cases, for a redox label, such as ferrocene, thedetector can be an electrode for amperometric assay of redox molecules.For example, if the redox label is present in a reduced form offerrocene, then the electrode at high electrode potential can provide anoxidation of the reduced form of ferrocene, thereby converting it to anoxidized form of ferrocene. The generated current can be proportional tothe concentration of ferrocene label in the solution.

In one embodiment, reporter nucleic acid or signal expansion nucleicacid can contain a fluorescent label and a quencher such that cleavedreporter nucleic acid provides a fluorescent signal and uncleavedreporter nucleic acid does not provide a fluorescent signal. In somecases, the reporter nucleic acid or signal expansion nucleic acid cancontain a label (e.g., a fluorescent label or an enzyme such as horseradish peroxidase) and can be attached to a solid support (e.g., a wellof a microtiter plate). For example, the reporter nucleic acid or signalexpansion nucleic acid can be attached to a solid support such thatcleavage at the amplifying restriction endonuclease cut site by theamplifying restriction endonuclease releases a portion of the reporternucleic acid or the signal expansion nucleic acid that contains thelabel. The resulting reaction mixture can be collected and assessed forthe presence, absence, or amount of released portions of the reporternucleic acid or signal expansion nucleic acid using the label. Forexample, the released portions of the reporter nucleic acid or thesignal expansion nucleic acid, if present, can be transferred from onewell of a microtiter plate (e.g., a 96-well plate) that contained thereporter nucleic acid or the signal expansion nucleic acid to anotherwell of a microtiter plate, where the transferred material can beassessed for a signal from the label. Any number of molecules of a labelcan be attached to one reporter nucleic acid molecule or one signalexpansion nucleic acid molecule. For example, a reporter nucleic acidmolecule or a single signal expansion nucleic acid molecule can containone, two, three, four, five, or more fluorescent molecules.

Any appropriate method can be used to attach a label to a nucleic acidcomponent of reporter nucleic acid or signal expansion nucleic acid. Insome cases, a label can be attached by an ionic or covalent attachment.For example, covalent bonds such as amide bonds, disulfide bonds, andthioether bonds, or bonds formed by crosslinking agents can be used. Insome cases, a non-covalent linkage can be used. The attachment can be adirect attachment or an indirect attachment. For example, a linker canbe used to attach a label to a nucleic acid component of reporternucleic acid or signal expansion nucleic acid. In some cases, nucleicacid can include a thiol modification, and a label can be conjugated tothe thiol-containing nucleic acid based on succinimidyl4-[N-maleimidomethyl]cyclo-hexane-1-carboxylate (SMCC) using techniquessimilar to those described elsewhere (Dill et al., Biosensors andBioelectronics, 20:736-742 (2004)). In some cases, a biotinylatednucleic acid and a streptavidin-containing label can be attached to oneanother via a biotin-streptavidin interaction. A label can be conjugatedwith streptavidin using, for example, sulfosuccinimidyl6-(3′-[2-pyridyldithio]-propionamido)hexanoate. A label can be attachedat any location of a nucleic acid component of reporter nucleic acid orsignal expansion nucleic acid. For example, a label can be attached atan end (e.g., a 5′ end or 3′ end) of a nucleic acid component, in themiddle of a nucleic acid component, or at any position along the lengthof a nucleic acid component of reporter nucleic acid or signal expansionnucleic acid.

The methods and materials provided herein can be used to detect targetnucleic acid in any type of sample. For example, blood samples, serumsamples, saliva samples, nasal swab samples, stool samples, urinesamples, tissue samples (e.g., tissue biopsy samples), environmentalsamples (e.g., water samples, soil samples, and air samples), foodsamples (e.g., meat samples, produce samples, or drink samples), andindustrial samples (e.g., air filter samples and samples collected fromwork stations) can be collected and assessed for target nucleic acid.Once obtained, a sample to be assessed can be processed to obtainnucleic acid. For example, a nucleic acid extraction can be performed ona tissue sample to obtain a sample that is enriched for nucleic acid. Insome cases, a sample can be heated or treated with a cell lysis agent torelease nucleic acid from cells present in the sample.

Once obtained, a sample to be assessed can be contacted with a probenucleic acid as described herein. This contacting step can be carriedout for any period of time and at any temperature that allows targetnucleic acid to hybridize with probe nucleic acid. For example, thisstep can be performed between 10 seconds and 24 hours (e.g., between 30seconds and 12 hours, between 30 seconds and 8 hours, between 30 secondsand 4 hours, between 30 seconds and 2 hours, between 30 seconds and 1hour, between 1 minute and 24 hours, between 1 minute and 12 hours,between 1 minute and 8 hours, between 1 minute and 4 hours, between 1minute and 2 hours, between 1 minute and 1 hour, between 5 minutes and 1hour, between 10 minutes and 1 hour, between 15 minutes and 1 hour, orbetween 30 minutes and 1 hour). The initial temperature can be between15° C. and 100° C. (e.g., between 23° C. and 98° C., between 23° C. and90° C., between 23° C. and 85° C., between 23° C. and 75° C., between23° C. and 65° C., between 23° C. and 55° C., between 23° C. and 45° C.,between 23° C. and 35° C., between 30° C. and 95° C., between 30° C. and85° C., between 30° C. and 75° C., between 30° C. and 65° C., between30° C. and 55° C., between 30° C. and 45° C., between 20° C. and 40° C.,between 20° C. and 30° C., and between 25° C. and 35° C.). Thetemperature during this contacting step can remain constant or can beincreased or decreased. For example, the initial temperature can bebetween about 40° C. and about 85° C., and then the temperature can beallowed to decrease to room temperature over a period of about 30seconds to about 30 minutes (e.g., between about 30 seconds and about 15minutes, between about 30 seconds and about 10 minutes, between about 1minute and about 30 minutes, between about 1 minute and about 15minutes, or between about 1 minute and about 5 minutes).

Contact of the sample (e.g., a sample to be tested or suspected tocontain target nucleic acid) with probe nucleic acid can occur in thepresence of the recognition restriction endonucleases, or a separatestep of adding the recognition restriction endonucleases to the reactioncan be performed. The recognition restriction endonuclease step can becarried out for any period of time and at any temperature that allowsthe recognition restriction endonuclease to cleave recognitionrestriction endonuclease cut sites formed by the hybridization of targetnucleic acid to the probe nucleic acid. For example, this step can beperformed between one second and 24 hours (e.g., between one second and30 minutes, between one second and one hour, between five seconds andone hour, between 30 seconds and 24 hours, between 30 seconds and 12hours, between 30 seconds and 8 hours, between 30 seconds and 4 hours,between 30 seconds and 2 hours, between 30 seconds and 1 hour, between 1minute and 24 hours, between 1 minute and 12 hours, between 1 minute and8 hours, between 1 minute and 4 hours, between 1 minute and 2 hours,between 1 minute and 1 hour, between 5 minutes and 1 hour, between 10minutes and 1 hour, between 15 minutes and 1 hour, or between 30 minutesand 1 hour). The temperature can be between 15° C. and 75° C. (e.g.,between 15° C. and 75° C., between 15° C. and 65° C., between 15° C. and55° C., between 15° C. and 45° C., between 15° C. and 35° C., between15° C. and 30° C., between 23° C. and 55° C., between 23° C. and 45° C.,between 30° C. and 65° C., between 30° C. and 55° C., between 30° C. and45° C., between 30° C. and 40° C., between 35° C. and 40° C., andbetween 36° C. and 38° C.). Any appropriate concentration of recognitionrestriction endonuclease can be used. For example, between about 0.001units and 1000 units (e.g., between about 0.001 units and 750 units,between about 0.001 units and 500 units, between about 0.001 units and250 units, between about 0.001 units and 200 units, between about 0.001units and 150 units, between about 0.001 units and 100 units, betweenabout 0.001 units and 50 units, between about 0.001 units and 25 units,between about 0.001 units and 10 units, between about 0.001 units and 1unit, between about 0.001 units and 0.1 units, between about 0.01 unitsand 1000 units, between about 0.1 units and 1000 units, between about 1unit and 1000 units, between about 10 units and 1000 units, betweenabout 50 units and 1000 units, between about 0.5 units and 100 units, orbetween about 1 unit and 100 units) of restriction endonuclease can beused. Other restriction endonuclease reaction conditions such as saltconditions can be used according to manufacture's instructions.

When one step of a method provided herein is completed, the resultingreaction product containing cleaved nucleic acid can be used in the nextstep. For example, cleaved nucleic acid of a reaction product can beremoved from uncleaved nucleic acid and used in the next step of themethod. For example, when probe nucleic acid is attached to a solidsupport, the released portions of probe nucleic acid that contain anamplifying restriction endonuclease can be collected and placed incontact with reporter nucleic acid or signal expansion nucleic acid asdescribed herein. The resulting reaction products of a particular stepcan be manually or automatically (e.g., robotically) transferred to alocation containing nucleic acid for the next step (e.g., reporternucleic acid or signal expansion nucleic acid), which nucleic acid canbe attached or not attached to a solid support. In some cases, onereaction of a method described herein can be carried out at one location(e.g., a chamber) of a microfluidic device or blister package device,and the reaction products that are generated can be moved to anotherlocation (e.g., another chamber) that contains nucleic acid for the nextstep (e.g., reporter nucleic acid or signal expansion nucleic acid) viaa channel. In some cases, cleaved nucleic acid of a reaction product canbe used in the next step of the method by removing the uncleaved nucleicacid from the reaction product. For example, when magnetic beads areused as a solid support, a magnetic force can be used to remove themagnetic beads and any attached uncleaved nucleic acid from the reactionproduct.

Any appropriate method can be used to detect cleaved reporter nucleicacid and/or signal expansion nucleic acid to determine the presence,absence, or amount of target nucleic acid in a sample. For example, sizeseparation techniques can be used to assess reaction products forcleaved reporter nucleic acid and/or signal expansion nucleic acid.Examples of such size separation techniques include, without limitation,gel electrophoresis and capillary electrophoresis techniques. In somecases, a melt curve analysis can be performed to assess reactionproducts for cleaved reporter nucleic acid and/or signal expansionnucleic acid. As described herein, a label can be used to aid in thedetection of cleaved nucleic acid (e.g., reporter nucleic acid and/orsignal expansion nucleic acid). Examples of labels that can be usedinclude, without limitation, fluorescent labels (with or without the useof quenchers), dyes, antibodies, radioactive material, enzymes (e.g.,horse radish peroxidase, alkaline phosphatese, laccase, galactosidase,or luciferase), redox labels (e.g., ferrocene redox labels), metallicparticles (e.g., gold nanoparticles), and green fluorescent proteinbased labels. For example, the release of fluorescently labeled portionsof reporter nucleic acid and/or signal expansion nucleic acid from asolid support can be assessed using common fluorescent label detectors.In some cases, cleaved reporter nucleic acid and/or signal expansionnucleic acid can be detected electrochemically. For electrochemicaldetection, the reporter nucleic acid and/or signal expansion nucleicacid can include a ferrocene redox label. Reporter nucleic acid and/orsignal expansion nucleic acid containing ferrocene can be obtained bycoupling ferrocene carboxylic acid with an amino-modifiedoligonucleotide using the carbodiimide reaction in the presence of anexcess of ferrocene carboxylic acid. In one embodiment, for a redoxlabel, such as ferrocene, the detector can be an electrode foramperometric assay of redox molecules. For example, if the redox labelis present in a reduced form of ferrocene, then the electrode at highelectrode potential can provide an oxidation of the reduced form offerrocene, thereby converting it to an oxidized form of ferrocene. Thegenerated current can be proportional to the concentration of ferrocenelabel in the solution.

The methods and materials provided herein can be used to assess one ormore samples for target nucleic acid in real-time. For example, afluorescent label/quencher system or an electrochemical redox labelsystem can be used to detect cleavage of reporter nucleic acid and/orsignal expansion nucleic acid in real time.

The methods and materials provided herein can be used to assess one ormore samples (e.g., two, three, four, five, six, seven, eight, nine,ten, 20, 50, 100, 500, 1000, or more) for a single type of targetnucleic acid. In some case, the methods and materials provided hereincan be used in a multiplex manner to assess one or more samples for morethan one (e.g., two, three, four, five, six, seven, eight, nine, ten,20, 50, 100, 500, 1000, or more) type of target nucleic acid. Forexample, target nucleic acid for ten different sequences (e.g., tendifferent sequences from a single bacterial species or strain, or adifferent sequence from ten different bacterial species or strains) canbe used to design ten different probe nucleic acid molecules. In suchcases, a different label can used to correspond to each probe nucleicacid such that the detected signals can indicate which of the ten targetnucleic acids are being detected.

This document also provides kits for performing the methods describedherein. For example, a kit provided herein can include probe nucleicacid with or without being attached to a solid support and/or reporternucleic acid with or without being attached to a solid support. In somecases, such a kit can include a recognition restriction endonuclease,first signal expansion nucleic acid, second signal expansion nucleicacid, or a combination thereof. In some cases, a kit can be configuredinto a microfluidic device that allows for the movement of probe nucleicacid, first signal expansion nucleic acid, second signal expansionnucleic acid, reporter nucleic acid, or recognition restrictionendonucleases (or any combination thereof) as well as a cleaved portionof any such nucleic acid in a manner that allows a detection methodprovided herein to be carried out with or without the nucleic acid beingattached to a solid support. For example, a kit provided herein can be amicrofluidic device capable of receiving a sample and contacting thatsample with probe nucleic acid. The probe nucleic acid can be designedto include a length of nucleotides followed by the sequencecomplementary to the target nucleic acid, which can create a recognitionrestriction endonuclease cut site, followed by an amplifying restrictionendonuclease. The distance from the recognition restriction endonucleasecut site to the amplifying restriction endonuclease can be relativelyshort (e.g., 100, 50, 25, 10, or less nucleotides), while the distancefrom the recognition restriction endonuclease cut site to the beginningof the length of nucleotides can be relatively long (e.g., 50, 100, 150,200, 500, 1000, 2000, or more). In such cases, cleavage of the probenucleic acid at the recognition restriction endonuclease cut site canresult in a relatively small portion that contains the amplifyingrestriction endonuclease and is capable of travelling faster than thelarger uncleaved probe nucleic acid. This difference can allow thecleaved portion containing the amplifying restriction endonuclease toreach an area of the microfluidic device containing signal expansionnucleic acid or reporter nucleic acid so that the next reaction can becarried out without the presence of uncleaved probe nucleic acid. Insome cases, after the smaller portion containing the amplifyingrestriction endonuclease enters the area containing signal expansionnucleic acid or reporter nucleic acid, a valve can be used to preventthe larger uncleaved probe nucleic acid from entering. In some cases, afilter can be used to limit the ability of larger uncleaved probenucleic acid from proceeding to the next reaction location. Similarapproaches can be used during other steps of a method provided herein toseparate cleaved nucleic acid from uncleaved nucleic acid.

In some cases, a kit provided herein can be a portable or self-containeddevice, packet, vessel, or container that can be used, for example, infield applications. For example, such a kit can be configured to allow auser to insert a sample for analysis. Once inserted, the sample can beheated (e.g., heated to about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,75, 80, 85, 90, 95, or more ° C.) and/or cooled by a heating or coolingmechanism located within the kit. For example, an exothermic orendothermic chemical reaction can be initiated within the kit toincrease, decrease, or maintain the temperature. Such exothermic orendothermic chemical reactions can be carried out within the kit withoutbeing in fluid communication with the reactions of the target nucleicacid detection method. An iron oxidation reaction is an example of anexothermic chemical reaction that can be used to heat a kit providedherein. An endothermic chemical reaction that can be used to cool a kitprovided herein can be a reaction that includes the use of ammoniumchloride and water, potassium chloride and water, or sodium carbonateand ethanoic acid. In general, when detecting DNA target nucleic acid,the kit can be designed to generate, if needed, enough heat to denaturedouble stranded DNA present within the sample. The kit also can bedesigned to generate appropriate heating and cooling temperatures tocarry out each step of a detection method provided herein. In somecases, a kit provided herein can include a temperature indicator (e.g.,color indicator or thermometer) to allows a user to assess temperature.

In some cases, a kit can be designed to provide a user with a “yes” or“no” indication about the presence of target nucleic acid within atested sample. For example, a label having the ability to generate achange in pH can be used, and a visual indicator (e.g., a pH-based colorindicator) can be used to inform the user of the presence of targetnucleic acid based on a change in pH.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Formation and Cleavage of Target-Probe Hybrids

An oligonucleotide probe (5′-thiol-GGT AGT GCG AAA TGC CAT TGC TAG TTGTTT-biotin-3′; SEQ ID NO:1) that was modified with a thiol group at the5′ end and a biotin molecule at the 3′ end was conjugated to horseradishperoxidase (HRP). Conjugation was performed using the SMCC reagentaccording to a technique modified from Dill et al. (Biosensors andBioelectronics, 20:736-742 (2004)). The HRP conjugate solution wasincubated with a streptavidin-coated ELISA plate to immobilize theHRP-oligonucleotide probe to the surface via a biotin-streptavidininteraction. The ELISA plate was then incubated with differentconcentrations of a target oligonucleotide (5′-AAA CAA CTA GCA ATG GCATTT-3′; SEQ ID NO:2). The target oligonucleotide sequence wasreverse-complementary to the probe sequence to form a double-strandedhybrid molecule. After washing, the plate was incubated in a solutioncontaining the restriction endonuclease BfaI. BfaI specificallyrecognizes the sequence 5′-CTAG-3′ and cleaves the double-stranded,target-probe hybrids to release the HRP-oligonucleotide into thereaction solution. After a two-hour incubation at 37° C., the reactionsolution was transferred to a new ELISA plate. The cleavedHRP-oligonucleotide was contacted to 3,3′,5,5′-tetramethyl benzidine(TMB) to form a colored reaction product.

When the restriction endonuclease BfaI was added in excess to thereaction mixture, a clear direct dependence between the amount ofreleased HRP-probe and the concentration of oligonucleotide target wasobserved (FIG. 6A). The detectable target concentration wasapproximately 1 nM. This detection limit was obtained by directmeasurement without any secondary signal amplification. The addition ofa restriction endonuclease signal amplification cascade as describedherein can further improve the detection limit by several orders ofmagnitude.

When the HRP-oligonucleotide probes were pre-incubated with an excess oftarget oligonucleotide (500 nM), the amount of cleavedHRP-oligonucleotide probe was limited by the amount of recognitionrestriction endonuclease BfaI (FIG. 6B). Taken together, these datademonstrate that recognition restriction endonucleases can be used toinitiate the restriction endonuclease cascades described herein.

Example 2 Detecting Target Nucleic Acid Using Probe Nucleic Acid andReporter Nucleic Acid

A target nucleic acid is selected. Once selected, target nucleic acid isanalyzed using a common genetic database such as GenBank® and/or acomputer-based sequence analysis program to identify a portion of thetarget nucleic acid that contains a cut site for a restrictionendonuclease. Probe nucleic acid is designed to be complementary to atleast a portion of target nucleic acid that contains a cut site. Oncedesigned and obtained by standard oligonucleotide synthesis methods,probe nucleic acid is conjugated to an amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A sample to be tested is incubated in the first well.If target nucleic acid is present in the sample, at least a portion ofthe target nucleic acid hybridizes to the probe nucleic acid, andthereby forms a recognition restriction endonuclease cut site. Therecognition restriction endonuclease is added to the first well havingthe sample and probe nucleic acid. The microtiter plate is incubated at37° C. for an appropriate length of time for the cleavage reaction toproceed.

Upon cleavage of probe nucleic acid by the recognition restrictionendonuclease, the reaction solution containing the released portion ofthe probe nucleic acid is transferred into a second well. The secondwell contains reporter nucleic acid that is immobilized to the surfaceand contains at least one double-stranded portion having an amplifyingrestriction endonuclease cut site. Reporter nucleic acid also has afluorescent label. Upon transfer to the second chamber, the amplifyingrestriction endonuclease bound to the released portion of the probenucleic acid contacts the reporter nucleic acid. The amplifyingrestriction endonuclease cleaves reporter nucleic acid at thedouble-stranded amplifying restriction endonuclease cut site to form atleast two portions. The liberated portion of the reporter nucleic acidhaving the fluorescent label is moved to a third microtiter plate well,and a standard fluorescent reader is used to measure any fluorescentsignal.

A standard curve of known amounts of target nucleic acid is used toquantify the amount of target nucleic acid in the tested sample.

Example 3 Detecting Target Nucleic Acid Using Probe Nucleic Acid, FirstSignal Expansion Nucleic Acid, Second Signal Expansion Nucleic Acid, andReporter Nucleic Acid

Once selected, target nucleic acid is analyzed using a common geneticdatabase such as GenBank® and/or a computer-based sequence analysisprogram to identify a portion of target nucleic acid that contains a cutsite for a restriction endonuclease. Probe nucleic acid is designedbased on the desired target nucleic acid as described herein. Standardoligonucleotide synthesis methods are used to make the probe nucleicacid, which is then conjugated to an initial amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A sample to be tested for the target nucleic acid isincubated in the first well. If target nucleic acid is present in thesample, at least a portion of target nucleic acid hybridizes to probenucleic acid and thereby forms a recognition restriction endonucleasecut site. Recognition restriction endonuclease is added to the firstwell having the sample and probe nucleic acid. The microtiter plate isincubated at 37° C. for an appropriate length of time for the cleavagereaction to proceed. After cleavage of the probe nucleic acid:targetnucleic acid hybrid by the recognition restriction endonuclease, thereaction solution containing the free portion of probe nucleic acid istransferred to another well that includes first signal expansion nucleicacid and second signal expansion nucleic acid. The first signalexpansion nucleic acid and second signal expansion nucleic acid createsa positive feedback loop that causes an exponential acceleration ofrelease of initial amplifying restriction enzymes. The reaction productfrom this well is transferred to another well containing reporternucleic acid, and cleavage of the reporter nucleic acid is used todetermine the presence, absence, or amount of target nucleic acid in thesample. A standard curve of known amounts of target nucleic acid is usedto quantify the amount of target nucleic acid in the tested sample.

Example 4 Detecting the Presence or Absence of Bacteria

The presence or absence of methicillin-resistant Staphylococcus aureus(MRSA) in a sample is detected using an enzymatic amplification cascade.A MRSA-specific target nucleic acid is analyzed using a common geneticdatabase such as GenBank and/or a computer-based sequence analysisprogram to identify a portion of target nucleic acid that contains a cutsite for a restriction endonuclease. Probe nucleic acid is designed tobe complementary to at least a portion of the selected target nucleicacid. Once designed and obtained by standard oligonucleotide synthesismethods, probe nucleic acid is conjugated to an amplifying restrictionendonuclease and immobilized to the surface of a first well of amicrotiter plate. A biological sample (e.g., tissue sample or nasalswab) that is suspected of having MRSA is obtained, and the nucleic acidfrom that sample is incubated in the first well. If MRSA is present inthe sample, at least a portion of the MRSA-specific nucleic acidhybridizes to the probe nucleic acid and thereby forms a recognitionrestriction endonuclease cut site. Recognition restriction endonucleaseis added to the first well having the sample and probe nucleic acid. Themicrotiter plate is incubated at 37° C. for an appropriate length oftime for the cleavage reaction to proceed.

After cleavage of the probe nucleic acid:target nucleic acid hybrid bythe recognition restriction endonuclease, the reaction solution in thefirst well is transferred to a second well containing reporter nucleicacid that is immobilized to the surface of the second well and that hasat least one double-stranded portion having an amplifying restrictionendonuclease cut site. The reporter nucleic acid also has a fluorescentlabel. In some cases, first signal expansion nucleic acid and secondsignal expansion nucleic acid are used prior to the report nucleic acidstep to increase the level of target nucleic acid detection. The firstsignal expansion nucleic acid and second signal expansion nucleic acidcan include labels in which case they can be used together with reporternucleic acid or in place of reporter nucleic acid.

After transferring the reaction mixture to the second chamber, theamplifying restriction endonucleases of the released portions of probenucleic acid contact reporter nucleic acid, and the microtiter plate isincubated at an appropriate temperature (e.g., at 37° C.) for anappropriate length of time for the cleavage reaction to proceed. Theamplifying restriction endonucleases cleave reporter nucleic acid at thedouble-stranded amplifying restriction endonuclease cut site to form atleast two portions. The reaction solution of the second well istransferred to a third well for fluorescence detection using afluorescent microtiter plate reader. Fluorescence in the third well isindicative of MRSA-specific nucleic acid present in the sample. If thesample was obtained from a patient, such a result is indicative of aMRSA infection in that patient. If no fluorescence is detected in thethird well, such a result is indicative of the absence of MRSA in thesample.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for assessing a sample for target nucleic acid, said method comprising: (a) contacting said sample with a probe nucleic acid comprising an amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of said target nucleic acid under conditions wherein, if said target nucleic acid is present in said sample, at least a portion of said target nucleic acid hybridizes to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site, (b) contacting said double-stranded portion of nucleic acid with a recognition restriction endonuclease having the ability to cut said double-stranded portion of nucleic acid at said restriction endonuclease cut site under conditions wherein said recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site, thereby separating a portion of said probe nucleic acid comprising an amplifying restriction endonuclease from at least another portion of said probe nucleic acid, (c) contacting said portion of said probe nucleic acid comprising an amplifying restriction endonuclease with a first nucleic acid comprising an amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said probe nucleic acid comprising an amplifying restriction endonuclease under conditions wherein the amplifying restriction endonuclease of said portion of said probe nucleic acid comprising an amplifying restriction endonuclease cleaves said first nucleic acid at said restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said probe nucleic acid comprising an amplifying restriction endonuclease, thereby separating a portion of said first nucleic acid comprising an amplifying restriction endonuclease from at least another portion of said first nucleic acid, (d) contacting said portion of said first nucleic acid comprising an amplifying restriction endonuclease with a reporter nucleic acid comprising a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said first nucleic acid comprising an amplifying restriction endonuclease under conditions wherein the amplifying restriction endonuclease of said portion of said first nucleic acid comprising an amplifying restriction endonuclease cleaves said reporter nucleic acid at said restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said first nucleic acid comprising an amplifying restriction endonuclease, thereby separating a portion of said reporter nucleic acid from at least another portion of said reporter nucleic acid, and (e) determining the presence or absence of said portion of said reporter nucleic acid, wherein the presence of said portion of said reporter nucleic acid indicates that said sample contains said target nucleic acid, and wherein the absence of said portion of said reporter nucleic acid indicates that said sample does not contain said target nucleic acid.
 2. The method of claim 1, wherein said probe nucleic acid comprises a first nucleic acid strand comprising said nucleotide sequence complementary to said sequence of said target nucleic acid hybridized to a second nucleic acid strand comprising said amplifying restriction endonuclease.
 3. The method of claim 1, wherein said probe nucleic acid is attached to a solid support.
 4. The method of claim 3, wherein said portion of said probe nucleic acid comprising an amplifying restriction endonuclease is released from said solid support via said step (b).
 5. The method of claim 1, wherein step (a) and step (b) are performed in the same compartment.
 6. The method of claim 1, wherein step (a) and step (b) are performed by adding said sample to a compartment comprising said probe nucleic acid and said recognition restriction endonuclease.
 7. The method of claim 1, wherein said method comprises using a plurality of said probe nucleic acid in said step (a).
 8. The method of claim 1, wherein said method comprises using a plurality of said reporter nucleic acid in said step (d).
 9. The method of claim 1, wherein said reporter nucleic acid in said step (d) is in molar excess of said portion of said first nucleic acid comprising an amplifying restriction endonuclease from said step (c).
 10. The method of claim 1, wherein said reporter nucleic acid is attached to a solid support.
 11. The method of claim 1, wherein said reporter nucleic acid comprises a single-stranded portion of nucleic acid.
 12. The method of claim 1, wherein said reporter nucleic acid comprises a label.
 13. The method of claim 12, wherein said label is a fluorescent label, a radioactive label, an enzyme label, or a redox label.
 14. The method of claim 12, wherein said portion of said reporter nucleic acid that is separated from said at least another portion of said reporter nucleic acid comprises said label.
 15. The method of claim 12, wherein said determining step (e) comprises detecting said label.
 16. The method of claim 1, wherein said steps (a), (b), (c), and (d) are performed without nucleic acid amplification, or wherein said steps (a), (b), (c), (d), and (e) are performed without nucleic acid amplification.
 17. The method of claim 1, wherein said determining step comprises determining the amount of said target nucleic acid present within said sample.
 18. The method of claim 1, wherein said first nucleic acid is attached to a solid support.
 19. The method of claim 1, wherein said first nucleic acid is directly attached to a solid support.
 20. The method of claim 18, wherein said portion of said first nucleic acid comprising an amplifying restriction endonuclease is released from said solid support via said step (c). 